Automated detection and repositioning of micro-objects in microfluidic devices

ABSTRACT

Methods are provided for the automated detection of micro-objects in a microfluidic device. In addition, methods are provided for repositioning micro-objects in a microfluidic device. In addition, methods are provided for separating micro-objects in a spatial region of the microfluidic device.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a non-provisional of, and thus claims the benefit ofand/or priority to, U.S. provisional patent application Ser. No.62/089,613, filed on Dec. 9, 2014 and U.S. provisional patentapplication Ser. No. 62/259,522, filed on Nov. 24, 2015, the entirecontents of which are incorporated herein by reference.

FIELD

The present invention generally relates to methods for detecting theresults of an assay within a microfluidic device. In particular, themethods can include steps for automatically selecting specific regionswithin the microfluidic device for detection of assay results.

BACKGROUND

Efficient and robust detection of micro-objects, such as biologicalcells or beads, on non-uniform or complicated backgrounds is crucial tothe automated manipulation of micro-objects in microfluidicenvironments. Due to the translucent appearance of certainmicro-objects, a non-uniform background that has features similar insize to such micro-objects creates significant detection challenges.Similarly, automated manipulation, such as repositioning cells, iscomplicated by specific features of OET technology. Some embodiments ofthe present invention are directed to the robust detection ofmicro-objects and re-positioning in microfluidic environments.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a method for theautomated detection of micro-objects disposed within a microfluidicdevice. The method comprises capturing a first image of a region in themicrofluidic device that may contain a micro-object of interest. Themethod further comprises inducing movement of fluid within said region.The method further comprises capturing a second image of said region.The method further comprises generating a differential image from thefirst and second images and identifying a micro-object of interest basedon the differential image.

In various embodiments, the region comprises one or more microfluidicdevice features that are captured in the first and second image, and thedifferential image does not contain the one or more microfluidic devicefeatures. In some embodiments, the one or more microfluidic devicefeatures include an array of phototransistors.

In various embodiments, the first and second images are captured using adigital camera or a CCD device.

In various embodiments, inducing movement of said fluid comprisesintroducing a discrete volume of fluid into said microfluidic device. Insome embodiments, the discrete volume of fluid is about 25 pL to about100 pL.

In various embodiments, the differential image is generated bysubtracting said first image from said second image, or vice versa. Insome embodiments, the method further comprises determining a first setof light intensity values for one or more pixels corresponding to thefirst image and a second set of light intensity value for one or morepixels corresponding to the second image and subtracting the first setof light intensity values from the second set of light intensity values,or vice versa, to generate a set of positive-value pixels and a set ofnegative-value pixels.

In some embodiments, the method further comprises analyzing the set ofpositive-value pixels to identify one or more sets of pixel clusters,wherein each pixel cluster comprises one or more pixels. The methodfurther comprises determining, for each of the one or more sets of pixelclusters, a feature set comprising information representing one or moreof: an area of the set of pixel clusters, a circumference of the set ofpixel clusters, a global morphology of the set of pixel clusters, alocal morphology of the set of pixel clusters, and a light intensityvalue associated with the set of pixel clusters. The method furthercomprises identifying, for each of the one or more sets of pixelclusters, whether the set of pixel clusters corresponds to amicro-object of interest, wherein the identification is based on thedetermined feature set for the set of pixel clusters.

In various embodiments, the method further comprises detecting pairs ofpositive-value and negative-value pixels or pixel clusters that differin their relative position by an amount consistent with said movement offluid induced in said region and identifying each such pair asrepresenting a current and former location, respectively, of themicro-object of interest.

In a second aspect, the present invention relates a method for theautomated detection of micro-objects disposed within a microfluidicdevice. The method comprises capturing with an imaging device a firstimage of a region of a microfluidic device that may contain amicro-object of interest. The method further comprises shifting saidmicrofluidic device relative to said imaging device. The method furthercomprises capturing with said imaging device a second image of theregion, wherein said second image is shifted relative to said firstimage. The method further comprises aligning said first image with saidsecond image. The method further comprises generating a differentialimage from said first and second images and identifying a micro-objectof interest based on the differential image.

In various embodiments, the region comprises one or more microfluidicdevice features that are captured in the first and second image, andwherein the differential image does not contain the one or moremicrofluidic device features. In some embodiments, the one or moremicrofluidic device features include an array of phototransistors.

In various embodiments, the imaging device is a digital camera or a CCDdevice.

In various embodiments, shifting said microfluidic device comprisesmoving a stage that is holding said microfluidic device in a directionperpendicular to an optical axis of said imaging device. In someembodiments, the microfluidic device is shifted by about 2 to about 3microns.

In various embodiments, the first and second images are alignedcomputationally, and wherein regions of said first and second imagesthat can't be aligned are discarded. In some embodiments, aligning saidfirst and second images comprises aligning circuit elements within themicrofluidic device.

In various embodiments, generating a differential image comprisessubtracting said first image from said second image, or vice versa. Insome embodiments, the method further comprises determining a first setof light intensity values for one or more pixels corresponding to thefirst image and a second set of light intensity value for one or morepixels corresponding to the second image and subtracting the first setof light intensity values from the second set of light intensity values,or vice versa, to generate a set of positive-value pixels and a set ofnegative-value pixels.

In some embodiments, the method further comprises analyzing the set ofpositive-value pixels to identify one or more sets of pixel clusters,wherein each pixel cluster comprises one or more pixels. The methodfurther comprises determining, for each set of pixel clusters of the oneor more sets of pixel clusters, a feature set comprising informationrepresenting one or more of: an area of the set of pixel clusters, acircumference of the set of pixel clusters, a global morphology of theset of pixel clusters, a local morphology of the set of pixel clusters,and a light intensity value associated with the set of pixel clusters.The method further comprises identifying, for each of the one or moresets of pixel clusters, whether the set of pixel clusters corresponds toa micro-object of interest, wherein the identification is based on thefeature set determined for the set of pixel clusters.

In some embodiments, the method further comprises detecting pairs ofpositive-value and negative-value pixels or pixel clusters that differin their relative position by an amount consistent with said movement ofsaid microfluidic device relative to said imaging device and identifyingeach such pair as representing a current and former location,respectively, of the micro-object of interest.

In a third aspect, the present invention provides a method for theautomated detection of micro-objects disposed within a microfluidicdevice. The method comprises capturing an image of a region in themicrofluidic device that may contain a micro-object of interest. Themethod further comprises determining periodic structures in the imageusing a Fourier transform. The method further comprises generating afiltered image by removing the period structures from the image andidentifying a micro-object of interest based on the filtered image.

In various embodiments, the periodic structures correspond to one ormore microfluidic device features. In some embodiments, the one or moremicrofluidic device features include an array of phototransistors.

In various embodiments, the method further comprises determining a setof light intensity values for one or more pixels corresponding to thefiltered image and generating a set of positive-value pixels based onthe filtered image.

In various embodiments, the method further comprises analyzing the setof positive-value pixels to identify one or more sets of pixel clusters,wherein each pixel cluster comprises one or more pixels. The methodfurther comprises determining, for each of the one or more sets ofpixels clusters, a feature set comprising information representing oneor more of: an area of the set of pixel clusters, a circumference of theset of pixel clusters, a global morphology of the set of pixel clusters,a local morphology of the set of pixel clusters and a light intensityvalue associated with the set of pixel clusters. The method furthercomprises identifying, for each of the one or more sets of pixelclusters, whether the set of pixel clusters corresponds to themicro-object of interest, wherein the identification is based on thefeature set determined for the set of pixel clusters.

In a fourth aspect, the present invention provides a method ofre-positioning micro-objects in a microfluidic device comprising a setof sequestration pens. The method comprises identifying a set ofmicro-objects disposed within the microfluidic device. The methodfurther comprises computing one or more trajectories, wherein eachtrajectory is a path that connects one micro-object of the set ofmicro-objects with one sequestration pen of the set of sequestrationpens. The method further comprises selecting, for one or moremicro-objects of the set of micro-objects, a trajectory of the one ormore trajectories. The method further comprises re-positioning at leastone micro-object of the one or more micro-objects of the set ofmicro-objects by moving the micro-object along its selected trajectory.

In various embodiments, the re-positioning at least one micro-object ofthe one or more micro-objects of the set of micro-objects comprisesmoving at least a first micro-object along its selected trajectory and asecond micro-object along its selected trajectory. In some embodiments,the first and second micro-objects are moved in parallel.

In various embodiments, the method further comprises computing a densityvalue associated with the set of micro-objects and computing the one ormore trajectories based, at least in part, on the density valueassociated with the set of micro-objects. In some embodiments, themethod further comprises determining that the density value exceeds athreshold value and computing, for at least one micro-object of the setof micro-objects, one or more trajectories connecting the micro-objectwith one or more sequestration pens of the set of sequestration pens. Insome embodiments, the method further comprises determining that thedensity value does not exceed a threshold value and computing, for atleast one sequestration pen of the set of sequestration pens, one ormore trajectories connecting the sequestration pen with one or moremicro-objects of the set of micro-objects.

In various embodiments, the method further comprises identifying the setof sequestration pens. In some embodiments, identifying the set ofsequestration pens comprises identifying empty sequestration pensamongst a plurality of sequestration pens.

In various embodiments, selecting a trajectory of the one or moretrajectories comprises selecting a trajectory for each micro-object thatis being repositioned such that the sum of the lengths of the selectedtrajectories is minimized. In some embodiments, minimizing the sum ofthe lengths of the selected trajectories comprises using at least one ofthe following: a greedy algorithm, a heuristics-based algorithm, anon-linear algorithm, and a constrained search.

In various embodiments, selecting a trajectory of the one or moretrajectories further comprises determining whether the trajectoryexceeds a pre-determined maximum length.

In some embodiments, re-positioning at least one micro-object of the oneor more micro-objects comprises accelerating each of the at least onemicro-objects from an initial velocity to a traveling velocity over afirst time period. In some embodiments, re-positioning at least onemicro-object of the one or more micro-objects comprises deceleratingeach of the at least one micro-objects from the traveling velocity to afinal velocity over a second time period.

In a fifth aspect, the present invention relates to a method ofre-positioning micro-objects in a microfluidic device. The methodcomprises identifying a set of micro-objects disposed within a specifiedspatial region of the microfluidic device. The method further comprisescalculating a set of vertices that divide the specified spatial regioninto sub-regions, each of which contains one or more micro-object(s) ofthe set of micro-objects. The method further comprises generating amodified first light cage for at least one micro-object of the set ofmicro-objects based on the calculated set of vertices; and moving themodified light cage relative to the specified spatial region of themicrofluidic device to re-position the at least one micro-object.

In various embodiments, the method further comprises computing, for afirst micro-object of the set of micro-objects, a first light cage. Themethod further comprises computing the intersection between the firstlight cage and the set of vertices. The method further comprisesgenerating the modified first light cage based on the intersectionbetween the first light cage and the set of vertices.

In various embodiments, the method further comprises calculating a setof vertices that maximize the distance between a subset of thecalculated set of vertices that are adjacent to each micro-object of theset of micro-objects and the micro-object.

In various embodiments, the method further comprises calculating a setof vertices that divide the specified spatial region into sub-regions,wherein at least a subset of the sub-regions contains a singlemicro-object of the set of micro-objects.

In various embodiments, the method further comprises calculating aDelaunay triangulation of the set of micro-objects. The method furthercomprises generating a Voronoi diagram based on the Delaunaytriangulation of the set of micro-objects and identifying the set ofvertices based on the Voronoi diagram.

In various embodiments, the method further comprises computing, for asecond micro-object of the set of micro-objects, a second light cage.The method further comprises computing the intersection between thesecond light cage and the set of vertices and generating a modifiedsecond light cage based on the intersection between the second lightcage and the set of vertices, wherein the modified second light cagedoes not intersect with the modified first light cage.

In various embodiments, the method further comprises moving both thefirst modified light cage and the second modified light cage relative tothe specified spatial region of the microfluidic device to physicallyseparate the first micro-object and the second micro-object. In someembodiments, the first micro-object and the second micro-object areinitially located in adjacent sub-regions of the specified spatialregion.

In various embodiments, the set of micro-objects is identified accordingto the methods described above with respect to the first aspect, secondaspect and third aspect of the present invention.

In a sixth aspect, the present invention relates to a machine readablestorage device for storing non-transitory machine readable instructionsfor carrying out the method of the first aspect of the presentinvention. In a seventh aspect, the present invention relates to amachine readable storage device for storing non-transitory machinereadable instructions for carrying out the method of the second aspectof the present invention. In an eighth aspect, the present inventionrelates to a machine readable storage device for storing non-transitorymachine readable instructions for carrying out the method of the thirdaspect of the present invention. In a ninth aspect, the presentinvention relates to a machine readable storage device for storingnon-transitory machine readable instructions for carrying out the methodof the fourth aspect of the present invention. In an tenth aspect, thepresent invention relates to a machine readable storage device forstoring non-transitory machine readable instructions for carrying outthe method of the fifth aspect of the present invention.

In various embodiments and aspects of the present invention, themicro-object of interest is a cell. In various embodiments and aspectsof the present invention, the cell is a mammalian cell. In variousembodiments and aspects of the present invention, the cell is selectedfrom the group consisting of a blood cell, a hybridoma, a cancer cell,and a transformed cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an example of a system for use with a microfluidicdevice and associated control equipment according to some embodiments ofthe invention.

FIGS. 2A and 2B illustrate a microfluidic device according to someembodiments of the invention.

FIGS. 2C and 2D illustrate sequestration pens according to someembodiments of the invention.

FIG. 2E illustrates a detailed sequestration pen according to someembodiments of the invention.

FIG. 2F illustrates a microfluidic device according to an embodiment ofthe invention.

FIG. 3A illustrates a specific example of a system for use with amicrofluidic device and associated control equipment according to someembodiments of the invention.

FIG. 3B illustrates an exemplary analog voltage divider circuitaccording to some embodiments of the invention.

FIG. 3C illustrates an exemplary GUI configured to plot temperature andwaveform data according to some embodiments of the invention.

FIG. 3D illustrates an imaging device according to some embodiments ofthe invention.

FIG. 3E illustrates communications between a motive module and a lightmodulating subsystem that control the projection of patterns of light ona microfluidic device, according to a specific embodiment of theinvention.

FIGS. 4A, 4B, and 4C depict the penning of micro-objects in parallel,according to one embodiment of the invention.

FIGS. 5A-5F illustrate the generation of modified light cages that canbe used to separate micro-objects, according to a specific embodiment ofthe present invention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

This specification describes exemplary embodiments and applications ofthe invention. The invention, however, is not limited to these exemplaryembodiments and applications or to the manner in which the exemplaryembodiments and applications operate or are described herein. Moreover,the figures may show simplified or partial views, and the dimensions ofelements in the figures may be exaggerated or otherwise not inproportion. In addition, as the terms “on,” “attached to,” “connectedto,” “coupled to,” or similar words are used herein, one element (e.g.,a material, a layer, a substrate, etc.) can be “on,” “attached to,”“connected to,” or “coupled to” another element regardless of whetherthe one element is directly on, attached to, connected to, or coupled tothe other element or there are one or more intervening elements betweenthe one element and the other element. In addition, where reference ismade to a list of elements (e.g., elements a, b, c), such reference isintended to include any one of the listed elements by itself, anycombination of less than all of the listed elements, and/or acombination of all of the listed elements.

Section divisions in the specification are for ease of review only anddo not limit any combination of elements discussed.

As used herein, “substantially” means sufficient to work for theintended purpose. The term “substantially” thus allows for minor,insignificant variations from an absolute or perfect state, dimension,measurement, result, or the like such as would be expected by a personof ordinary skill in the field but that do not appreciably affectoverall performance. When used with respect to numerical values orparameters or characteristics that can be expressed as numerical values,“substantially” means within ten percent.

As used herein, the term “ones” means more than one. As used herein, theterm “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

As used herein, the term “disposed” encompasses within its meaning“located.”

As used herein, a “microfluidic device” or “microfluidic apparatus” is adevice that includes one or more discrete microfluidic circuitsconfigured to hold a fluid, each microfluidic circuit comprised offluidically interconnected circuit elements, including but not limitedto region(s), flow path(s), channel(s), chamber(s), and/or pen(s), andat least two ports configured to allow the fluid (and, optionally,micro-objects suspended in the fluid) to flow into and/or out of themicrofluidic device. Typically, a microfluidic circuit of a microfluidicdevice will include at least one microfluidic channel and at least onechamber, and will hold a volume of fluid of less than about 1 mL, e.g.,less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9,8, 7, 6, 5, 4, 3, or 2 μL. In certain embodiments, the microfluidiccircuit holds about 1-2, 1-3, 1-4, 1-5, 2-5, 2-8, 2-10, 2-12, 2-15,2-20, 5-20, 5-30, 5-40, 5-50, 10-50, 10-75, 10-100, 20-100, 20-150,20-200, 50-200, 50-250, or 50-300 μL.

As used herein, a “nanofluidic device” or “nanofluidic apparatus” is atype of microfluidic device having a microfluidic circuit that containsat least one circuit element configured to hold a volume of fluid ofless than about 1 μL, e.g., less than about 750, 500, 250, 200, 150,100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL or less.Typically, a nanofluidic device will comprise a plurality of circuitelements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75,100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000,2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, ormore). In certain embodiments, one or more (e.g., all) of the at leastone circuit elements is configured to hold a volume of fluid of about100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pLto 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, oneor more (e.g., all) of the at least one circuit elements is configuredto hold a volume of fluid of about 100 to 200 nL, 100 to 300 nL, 100 to400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500 nL, 200to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to 600 nL,or 250 to 750 nL.

A “microfluidic channel” or “flow channel” as used herein refers to flowregion of a microfluidic device having a length that is significantlylonger than both the horizontal and vertical dimensions. For example,the flow channel can be at least 5 times the length of either thehorizontal or vertical dimension, e.g., at least 10 times the length, atleast 25 times the length, at least 100 times the length, at least 200times the length, at least 500 times the length, at least 1,000 timesthe length, at least 5,000 times the length, or longer. In someembodiments, the length of a flow channel is in the range of from about100,000 microns to about 500,000 microns, including any rangetherebetween. In some embodiments, the horizontal dimension is in therange of from about 100 microns to about 1000 microns (e.g., about 150to about 500 microns) and the vertical dimension is in the range of fromabout 25 microns to about 200 microns, e.g., from about 40 to about 150microns. It is noted that a flow channel may have a variety of differentspatial configurations in a microfluidic device, and thus is notrestricted to a perfectly linear element. For example, a flow channelmay be, or include one or more sections having, the followingconfigurations: curve, bend, spiral, incline, decline, fork (e.g.,multiple different flow paths), and any combination thereof. Inaddition, a flow channel may have different cross-sectional areas alongits path, widening and constricting to provide a desired fluid flowtherein.

As used herein, the term “obstruction” refers generally to a bump orsimilar type of structure that is sufficiently large so as to partially(but not completely) impede movement of target micro-objects between twodifferent regions or circuit elements in a microfluidic device. The twodifferent regions/circuit elements can be, for example, a microfluidicsequestration pen and a microfluidic channel, or a connection region andan isolation region of a microfluidic sequestration pen.

As used herein, the term “constriction” refers generally to a narrowingof a width of a circuit element (or an interface between two circuitelements) in a microfluidic device. The constriction can be located, forexample, at the interface between a microfluidic sequestration pen and amicrofluidic channel, or at the interface between an isolation regionand a connection region of a microfluidic sequestration pen.

As used herein, the term “transparent” refers to a material which allowsvisible light to pass through without substantially altering the lightas is passes through.

As used herein, the term “micro-object” refers generally to anymicroscopic object that may be isolated and collected in accordance withthe present invention. Non-limiting examples of micro-objects include:inanimate micro-objects such as microparticles; microbeads (e.g.,polystyrene beads, Luminex™ beads, or the like); magnetic beads;microrods; microwires; quantum dots, and the like; biologicalmicro-objects such as cells (e.g., embryos, oocytes, sperm cells, cellsdissociated from a tissue, eukaryotic cells, protist cells, animalcells, mammalian cells, human cells, immunological cells, hybridomas,cultured cells, cells from a cell line, cancer cells, infected cells,transfected and/or transformed cells, reporter cells, prokaryotic cell,and the like); biological organelles; vesicles, or complexes; syntheticvesicles; liposomes (e.g., synthetic or derived from membranepreparations); lipid nanorafts (as described in Ritchie et al. (2009)“Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs,”Methods Enzymol., 464:211-231), and the like; or a combination ofinanimate micro-objects and biological micro-objects (e.g., microbeadsattached to cells, liposome-coated micro-beads, liposome-coated magneticbeads, or the like). Beads may further have other moieties/moleculescovalently or non-covalently attached, such as fluorescent labels,proteins, small molecule signaling moieties, antigens, orchemical/biological species capable of use in an assay.

As used herein, the term “maintaining (a) cell(s)” refers to providingan environment comprising both fluidic and gaseous components and,optionally a surface, that provides the conditions necessary to keep thecells viable and/or expanding.

A “component” of a fluidic medium is any chemical or biochemicalmolecule present in the medium, including solvent molecules, ions, smallmolecules, antibiotics, nucleotides and nucleosides, nucleic acids,amino acids, peptides, proteins, sugars, carbohydrates, lipids, fattyacids, cholesterol, metabolites, or the like.

As used herein in reference to a fluidic medium, “diffuse” and“diffusion” refer to thermodynamic movement of a component of thefluidic medium down a concentration gradient.

The phrase “flow of a medium” means bulk movement of a fluidic mediumprimarily due to any mechanism other than diffusion. For example, flowof a medium can involve movement of the fluidic medium from one point toanother point due to a pressure differential between the points. Suchflow can include a continuous, pulsed, periodic, random, intermittent,or reciprocating flow of the liquid, or any combination thereof. Whenone fluidic medium flows into another fluidic medium, turbulence andmixing of the media can result.

The phrase “substantially no flow” refers to a rate of flow of a fluidicmedium that, averaged over time, is less than the rate of diffusion ofcomponents of a material (e.g., an analyte of interest) into or withinthe fluidic medium. The rate of diffusion of components of such amaterial can depend on, for example, temperature, the size of thecomponents, and the strength of interactions between the components andthe fluidic medium.

As used herein in reference to different regions within a microfluidicdevice, the phrase “fluidically connected” means that, when thedifferent regions are substantially filled with fluid, such as fluidicmedia, the fluid in each of the regions is connected so as to form asingle body of fluid. This does not mean that the fluids (or fluidicmedia) in the different regions are necessarily identical incomposition. Rather, the fluids in different fluidically connectedregions of a microfluidic device can have different compositions (e.g.,different concentrations of solutes, such as proteins, carbohydrates,ions, or other molecules) which are in flux as solutes move down theirrespective concentration gradients and/or fluids flow through thedevice.

A microfluidic (or nanofluidic) device can comprise “swept” regions and“unswept” regions. As used herein, a “swept” region is comprised of oneor more fluidically interconnected circuit elements of a microfluidiccircuit, each of which experiences a flow of medium when fluid isflowing through the microfluidic circuit. The circuit elements of aswept region can include, for example, regions, channels, and all orparts of chambers. As used herein, an “unswept” region is comprised ofone or more fluidically interconnected circuit element of a microfluidiccircuit, each of which experiences substantially no flux of fluid whenfluid is flowing through the microfluidic circuit. An unswept region canbe fluidically connected to a swept region, provided the fluidicconnections are structured to enable diffusion but substantially no flowof media between the swept region and the unswept region. Themicrofluidic device can thus be structured to substantially isolate anunswept region from a flow of medium in a swept region, while enablingsubstantially only diffusive fluidic communication between the sweptregion and the unswept region. For example, a flow channel of amicro-fluidic device is an example of a swept region while an isolationregion (described in further detail below) of a microfluidic device isan example of an unswept region.

As used herein, a “flow path” refers to one or more fluidicallyconnected circuit elements (e.g. channel(s), region(s), chamber(s) andthe like) that define, and are subject to, the trajectory of a flow ofmedium. A flow path is thus an example of a swept region of amicrofluidic device. Other circuit elements (e.g., unswept regions) maybe fluidically connected with the circuit elements that comprise theflow path without being subject to the flow of medium in the flow path.

The capability of biological micro-objects (e.g., biological cells) toproduce specific biological materials (e.g., proteins, such asantibodies) can be assayed in such a microfluidic device. In a specificembodiment of an assay, sample material comprising biologicalmicro-objects (e.g., cells) to be assayed for production of an analyteof interest can be loaded into a swept region of the microfluidicdevice. Ones of the biological micro-objects (e.g., mammalian cells,such as human cells) can be selected for particular characteristics anddisposed in unswept regions. The remaining sample material can then beflowed out of the swept region and an assay material flowed into theswept region. Because the selected biological micro-objects are inunswept regions, the selected biological micro-objects are notsubstantially affected by the flowing out of the remaining samplematerial or the flowing in of the assay material. The selectedbiological micro-objects can be allowed to produce the analyte ofinterest, which can diffuse from the unswept regions into the sweptregion, where the analyte of interest can react with the assay materialto produce localized detectable reactions, each of which can becorrelated to a particular unswept region. Any unswept region associatedwith a detected reaction can be analyzed to determine which, if any, ofthe biological micro-objects in the unswept region are sufficientproducers of the analyte of interest.

Microfluidic Devices and Systems for Operating and Observing SuchDevices.

FIG. 1 illustrates an example of a microfluidic device 100 and a system150 which can be used in the practice of the present invention. Aperspective view of the microfluidic device 100 is shown having apartial cut-away of its cover 110 to provide a partial view into themicrofluidic device 100. The microfluidic device 100 generally comprisesa microfluidic circuit 120 comprising a flow path 106 through which afluidic medium 180 can flow, optionally carrying one or moremicro-objects (not shown) into and/or through the microfluidic circuit120. Although a single microfluidic circuit 120 is illustrated in FIG.1, suitable microfluidic devices can include a plurality (e.g., 2 or 3)of such microfluidic circuits. Regardless, the microfluidic device 100can be configured to be a nanofluidic device. In the embodimentillustrated in FIG. 1, the microfluidic circuit 120 comprises aplurality of microfluidic sequestration pens 124, 126, 128, and 130,each having one or more openings in fluidic communication with flow path106. As discussed further below, the microfluidic sequestration penscomprise various features and structures that have been optimized forretaining micro-objects in the microfluidic device, such as microfluidicdevice 100, even when a medium 180 is flowing through the flow path 106.Before turning to the foregoing, however, a brief description ofmicrofluidic device 100 and system 150 is provided.

As generally illustrated in FIG. 1, the microfluidic circuit 120 isdefined by an enclosure 102. Although the enclosure 102 can bephysically structured in different configurations, in the example shownin FIG. 1 the enclosure 102 is depicted as comprising a supportstructure 104 (e.g., a base), a microfluidic circuit structure 108, anda cover 110. The support structure 104, microfluidic circuit structure108, and cover 110 can be attached to each other. For example, themicrofluidic circuit structure 108 can be disposed on an inner surface109 of the support structure 104, and the cover 110 can be disposed overthe microfluidic circuit structure 108. Together with the supportstructure 104 and cover 110, the microfluidic circuit structure 108 candefine the elements of the microfluidic circuit 120.

The support structure 104 can be at the bottom and the cover 110 at thetop of the microfluidic circuit 120 as illustrated in FIG. 1.Alternatively, the support structure 104 and the cover 110 can beconfigured in other orientations. For example, the support structure 104can be at the top and the cover 110 at the bottom of the microfluidiccircuit 120. Regardless, there can be one or more ports 107 eachcomprising a passage into or out of the enclosure 102. Examples of apassage include a valve, a gate, a pass-through hole, or the like. Asillustrated, port 107 is a pass-through hole created by a gap in themicrofluidic circuit structure 108. However, the port 107 can besituated in other components of the enclosure 102, such as the cover110. Only one port 107 is illustrated in FIG. 1 but the microfluidiccircuit 120 can have two or more ports 107. For example, there can be afirst port 107 that functions as an inlet for fluid entering themicrofluidic circuit 120, and there can be a second port 107 thatfunctions as an outlet for fluid exiting the microfluidic circuit 120.Whether a port 107 function as an inlet or an outlet can depend upon thedirection that fluid flows through flow path 106.

The support structure 104 can comprise one or more electrodes (notshown) and a substrate or a plurality of interconnected substrates. Forexample, the support structure 104 can comprise one or moresemiconductor substrates, each of which is electrically connected to anelectrode (e.g., all or a subset of the semiconductor substrates can beelectrically connected to a single electrode). The support structure 104can further comprise a printed circuit board assembly (“PCBA”). Forexample, the semiconductor substrate(s) can be mounted on a PCBA.

The microfluidic circuit structure 108 can define circuit elements ofthe microfluidic circuit 120. Such circuit elements can comprise spacesor regions that can be fluidly interconnected when microfluidic circuit120 is filled with fluid, such as flow channels, chambers, pens, traps,and the like. In the microfluidic circuit 120 illustrated in FIG. 1, themicrofluidic circuit structure 108 comprises a frame 114 and amicrofluidic circuit material 116. The frame 114 can partially orcompletely enclose the microfluidic circuit material 116. The frame 114can be, for example, a relatively rigid structure substantiallysurrounding the microfluidic circuit material 116. For example the frame114 can comprise a metal material.

The microfluidic circuit material 116 can be patterned with cavities orthe like to define circuit elements and interconnections of themicrofluidic circuit 120. The microfluidic circuit material 116 cancomprise a flexible material, such as a flexible polymer (e.g. rubber,plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), or thelike), which can be gas permeable. Other examples of materials that cancompose microfluidic circuit material 116 include molded glass, anetchable material such as silicone (e.g. photo-patternable silicone or“PPS”), photo-resist (e.g., SU8), or the like. In some embodiments, suchmaterials—and thus the microfluidic circuit material 116—can be rigidand/or substantially impermeable to gas. Regardless, microfluidiccircuit material 116 can be disposed on the support structure 104 andinside the frame 114.

The cover 110 can be an integral part of the frame 114 and/or themicrofluidic circuit material 116. Alternatively, the cover 110 can be astructurally distinct element, as illustrated in FIG. 1. The cover 110can comprise the same or different materials than the frame 114 and/orthe microfluidic circuit material 116. Similarly, the support structure104 can be a separate structure from the frame 114 or microfluidiccircuit material 116 as illustrated, or an integral part of the frame114 or microfluidic circuit material 116. Likewise the frame 114 andmicrofluidic circuit material 116 can be separate structures as shown inFIG. 1 or integral portions of the same structure.

In some embodiments, the cover 110 can comprise a rigid material. Therigid material may be glass or a material with similar properties. Insome embodiments, the cover 110 can comprise a deformable material. Thedeformable material can be a polymer, such as PDMS. In some embodiments,the cover 110 can comprise both rigid and deformable materials. Forexample, one or more portions of cover 110 (e.g., one or more portionspositioned over sequestration pens 124, 126, 128, 130) can comprise adeformable material that interfaces with rigid materials of the cover110. In some embodiments, the cover 110 can further include one or moreelectrodes. The one or more electrodes can comprise a conductive oxide,such as indium-tin-oxide (ITO), which may be coated on glass or asimilarly insulating material. Alternatively, the one or more electrodescan be flexible electrodes, such as single-walled nanotubes,multi-walled nanotubes, nanowires, clusters of electrically conductivenanoparticles, or combinations thereof, embedded in a deformablematerial, such as a polymer (e.g., PDMS). Flexible electrodes that canbe used in microfluidic devices have been described, for example, inU.S. 2012/0325665 (Chiou et al.), the contents of which are incorporatedherein by reference. In some embodiments, the cover 110 can be modified(e.g., by conditioning all or part of a surface that faces inward towardthe microfluidic circuit 120) to support cell adhesion, viability and/orgrowth. The modification may include a coating of a synthetic or naturalpolymer. In some embodiments, the cover 110 and/or the support structure104 can be transparent to light. The cover 110 may also include at leastone material that is gas permeable (e.g., PDMS or PPS).

FIG. 1 also shows a system 150 for operating and controllingmicrofluidic devices, such as microfluidic device 100. System 150, asillustrated, includes an electrical power source 192, an imaging device194, and a tilting device 190.

The electrical power source 192 can provide electric power to themicrofluidic device 100 and/or tilting device 190, providing biasingvoltages or currents as needed. The electrical power source 192 can, forexample, comprise one or more alternating current (AC) and/or directcurrent (DC) voltage or current sources. The imaging device 194 cancomprise a device, such as a digital camera, for capturing images insidemicrofluidic circuit 120. In some instances, the imaging device 194further comprises a detector having a fast frame rate and/or highsensitivity (e.g. for low light applications). The imaging device 194can also include a mechanism for directing stimulating radiation and/orlight beams into the microfluidic circuit 120 and collecting radiationand/or light beams reflected or emitted from the microfluidic circuit120 (or micro-objects contained therein). The emitted light beams may bein the visible spectrum and may, e.g., include fluorescent emissions.The reflected light beams may include reflected emissions originatingfrom an LED or a wide spectrum lamp, such as a mercury lamp (e.g. a highpressure mercury lamp) or a Xenon arc lamp. As discussed with respect toFIG. 3, the imaging device 194 may further include a microscope (or anoptical train), which may or may not include an eyepiece.

System 150 further comprises a tilting device 190 configured to rotate amicrofluidic device 100 about one or more axes of rotation. In someembodiments, the tilting device 190 is configured to support and/or holdthe enclosure 102 comprising the microfluidic circuit 120 about at leastone axis such that the microfluidic device 100 (and thus themicrofluidic circuit 120) can be held in a level orientation (i.e. at 0°relative to x- and y-axes), a vertical orientation (i.e. at 90° relativeto the x-axis and/or the y-axis), or any orientation therebetween. Theorientation of the microfluidic device 100 (and the microfluidic circuit120) relative to an axis is referred to herein as the “tilt” of themicrofluidic device 100 (and the microfluidic circuit 120). For example,the tilting device 190 can tilt the microfluidic device 100 at 0.1°,0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°,15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°,90° relative to the x-axis or any degree therebetween. The levelorientation (and thus the x- and y-axes) is defined as normal to avertical axis defined by the force of gravity. The tilting device canalso tilt the microfluidic device 100 (and the microfluidic circuit 120)to any degree greater than 90° relative to the x-axis and/or y-axis, ortilt the microfluidic device 100 (and the microfluidic circuit 120) 180°relative to the x-axis or the y-axis in order to fully invert themicrofluidic device 100 (and the microfluidic circuit 120). Similarly,in some embodiments, the tilting device 190 tilts the microfluidicdevice 100 (and the microfluidic circuit 120) about an axis of rotationdefined by flow path 106 or some other portion of microfluidic circuit120.

In some instances, the microfluidic device 100 is tilted into a verticalorientation such that the flow path 106 is positioned above or below oneor more sequestration pens. The term “above” as used herein denotes thatthe flow path 106 is positioned higher than the one or moresequestration pens on a vertical axis defined by the force of gravity(i.e. an object in a sequestration pen above a flow path 106 would havea higher gravitational potential energy than an object in the flowpath). The term “below” as used herein denotes that the flow path 106 ispositioned lower than the one or more sequestration pens on a verticalaxis defined by the force of gravity (i.e. an object in a sequestrationpen below a flow path 106 would have a lower gravitational potentialenergy than an object in the flow path).

In some instances, the tilting device 190 tilts the microfluidic device100 about an axis that is parallel to the flow path 106. Moreover, themicrofluidic device 100 can be tilted to an angle of less than 90° suchthat the flow path 106 is located above or below one or moresequestration pens without being located directly above or below thesequestration pens. In other instances, the tilting device 190 tilts themicrofluidic device 100 about an axis perpendicular to the flow path106. In still other instances, the tilting device 190 tilts themicrofluidic device 100 about an axis that is neither parallel norperpendicular to the flow path 106.

System 150 can further include a media source 178. The media source 178(e.g., a container, reservoir, or the like) can comprise multiplesections or containers, each for holding a different fluidic medium 180.Thus, the media source 178 can be a device that is outside of andseparate from the microfluidic device 100, as illustrated in FIG. 1.Alternatively, the media source 178 can be located in whole or in partinside the enclosure 102 of the microfluidic device 100. For example,the media source 178 can comprise reservoirs that are part of themicrofluidic device 100.

FIG. 1 also illustrates simplified block diagram depictions of examplesof control and monitoring equipment 152 that constitute part of system150 and can be utilized in conjunction with a microfluidic device 100.As shown, examples of such control and monitoring equipment 152 includea master controller 154 comprising a media module 160 for controllingthe media source 178, a motive module 162 for controlling movementand/or selection of micro-objects (not shown) and/or medium (e.g.,droplets of medium) in the microfluidic circuit 120, an imaging module164 for controlling an imaging device 194 (e.g., a camera, microscope,light source or any combination thereof) for capturing images (e.g.,digital images), and a tilting module 166 for controlling a tiltingdevice 190. The control equipment 152 can also include other modules 168for controlling, monitoring, or performing other functions with respectto the microfluidic device 100. As shown, the equipment 152 can furtherinclude a display device 170 and an input/output device 172.

The master controller 154 can comprise a control module 156 and adigital memory 158. The control module 156 can comprise, for example, adigital processor configured to operate in accordance with machineexecutable instructions (e.g., software, firmware, source code, or thelike) stored as non-transitory data or signals in the memory 158.Alternatively or in addition, the control module 156 can comprisehardwired digital circuitry and/or analog circuitry. The media module160, motive module 162, imaging module 164, tilting module 166, and/orother modules 168 can be similarly configured. Thus, functions,processes acts, actions, or steps of a process discussed herein as beingperformed with respect to the microfluidic device 100 or any othermicrofluidic apparatus can be performed by any one or more of the mastercontroller 154, media module 160, motive module 162, imaging module 164,tilting module 166, and/or other modules 168 configured as discussedabove. Similarly, the master controller 154, media module 160, motivemodule 162, imaging module 164, tilting module 166, and/or other modules168 may be communicatively coupled to transmit and receive data used inany function, process, act, action or step discussed herein.

The media module 160 controls the media source 178. For example, themedia module 160 can control the media source 178 to input a selectedfluidic medium 180 into the enclosure 102 (e.g., through an inlet port107). The media module 160 can also control removal of media from theenclosure 102 (e.g., through an outlet port (not shown)). One or moremedia can thus be selectively input into and removed from themicrofluidic circuit 120. The media module 160 can also control the flowof fluidic medium 180 in the flow path 106 inside the microfluidiccircuit 120. For example, in some embodiments media module 160 stops theflow of media 180 in the flow path 106 and through the enclosure 102prior to the tilting module 166 causing the tilting device 190 to tiltthe microfluidic device 100 to a desired angle of incline.

The motive module 162 can be configured to control selection, trapping,and movement of micro-objects (not shown) in the microfluidic circuit120. As discussed below with respect to FIGS. 2A and 2B, the enclosure102 can comprise a dielectrophoresis (DEP), optoelectronic tweezers(OET) and/or opto-electrowetting (OEW) configuration (not shown in FIG.1), and the motive module 162 can control the activation of electrodesand/or transistors (e.g., phototransistors) to select and movemicro-objects (not shown) and/or droplets of medium (not shown) in theflow path 106 and/or sequestration pens 124, 126, 128, 130.

The imaging module 164 can control the imaging device 194. For example,the imaging module 164 can receive and process image data from theimaging device 194. Image data from the imaging device 194 can compriseany type of information captured by the imaging device 194 (e.g., thepresence or absence of micro-objects, droplets of medium, accumulationof label, such as fluorescent label, etc.). Using the informationcaptured by the imaging device 194, the imaging module 164 can furthercalculate the position of objects (e.g., micro-objects, droplets ofmedium) and/or the rate of motion of such objects within themicrofluidic device 100.

The tilting module 166 can control the tilting motions of tilting device190. Alternatively or in addition, the tilting module 166 can controlthe tilting rate and timing to optimize transfer of micro-objects to theone or more sequestration pens via gravitational forces. The tiltingmodule 166 is communicatively coupled with the imaging module 164 toreceive data describing the motion of micro-objects and/or droplets ofmedium in the microfluidic circuit 120. Using this data, the tiltingmodule 166 may adjust the tilt of the microfluidic circuit 120 in orderto adjust the rate at which micro-objects and/or droplets of medium movein the microfluidic circuit 120. The tilting module 166 may also usethis data to iteratively adjust the position of a micro-object and/ordroplet of medium in the microfluidic circuit 120.

In the example shown in FIG. 1, the microfluidic circuit 120 isillustrated as comprising a microfluidic channel 122 and sequestrationpens 124, 126, 128, 130. Each pen comprises an opening to channel 122,but otherwise is enclosed such that the pens can substantially isolatemicro-objects inside the pen from fluidic medium 180 and/ormicro-objects in the flow path 106 of channel 122 or in other pens. Insome instances, pens 124, 126, 128, 130 are configured to physicallycorral one or more micro-objects within the microfluidic circuit 120.Sequestration pens in accordance with the present invention can comprisevarious shapes, surfaces and features that are optimized for use withDEP, OET, OEW, and/or gravitational forces, as will be discussed andshown in detail below.

The microfluidic circuit 120 may comprise any number of microfluidicsequestration pens. Although five sequestration pens are shown,microfluidic circuit 120 may have fewer or more sequestration pens. Asshown, microfluidic sequestration pens 124, 126, 128, and 130 ofmicrofluidic circuit 120 each comprise differing features and shapeswhich may provide one or more benefits useful in performing assays (e.g.culturing and retaining micro-objects used in assays). In someembodiments, the microfluidic circuit 120 comprises a plurality ofidentical microfluidic sequestration pens. In some embodiments, themicrofluidic circuit 120 comprises a plurality of microfluidicsequestration pens, wherein two or more of the sequestration penscomprise differing structures and/or features. For example, thesequestration pens can provide differing benefits with regard toperforming assays.

In the embodiment illustrated in FIG. 1, a single channel 122 and flowpath 106 is shown. However, other embodiments may contain multiplechannels 122, each configured to comprise a flow path 106. Themicrofluidic circuit 120 further comprises an inlet valve or port 107 influid communication with the flow path 106 and fluidic medium 180,whereby fluidic medium 180 can access channel 122 via the inlet port107. In some instances, the flow path 106 comprises a single path. Insome instances, the single path is arranged in a zigzag pattern wherebythe flow path 106 travels across the microfluidic device 100 two or moretimes in alternating directions.

In some instances, microfluidic circuit 120 comprises a plurality ofparallel channels 122 and flow paths 106, wherein the fluidic medium 180within each flow path 106 flows in the same direction. In someinstances, the fluidic medium within each flow path 106 flows in atleast one of a forward or reverse direction. In some instances, aplurality of sequestration pens are configured (e.g., relative to achannel 122) such that they can be loaded with target micro-objects inparallel.

In some embodiments, microfluidic circuit 120 further comprises one ormore micro-object traps 132. The traps 132 are generally formed in awall forming the boundary of a channel 122, and may be positionedopposite an opening of one or more of the microfluidic sequestrationpens 124, 126, 128, 130. In some embodiments, the traps 132 areconfigured to receive or capture a single micro-object from the flowpath 106. In some embodiments, the traps 132 are configured to receiveor capture a plurality of micro-objects from the flow path 106. In someinstances, the traps 132 comprise a volume approximately equal to thevolume of a single target micro-object.

The traps 132 may further comprise an opening which is configured toassist the flow of targeted micro-objects into the traps 132. In someinstances, the traps 132 comprise an opening having a height and widththat is approximately equal to the dimensions of a single targetmicro-object, whereby larger micro-objects are prevented from enteringinto the micro-object trap. The traps 132 may further comprise otherfeatures configured to assist in retention of targeted micro-objectswithin the trap 132. In some instances, the trap 132 is aligned with andsituated on the opposite side of a channel 122 relative to the openingof a microfluidic sequestration pen, such that upon tilting themicrofluidic device 100 about an axis parallel to the channel 122, thetrapped micro-object exits the trap 132 at a trajectory that causes themicro-object to fall into the opening of the sequestration pen. In someinstances, the trap 132 comprises a side passage 134 that is smallerthan the target micro-object in order to facilitate flow through thetrap 132 and thereby increase the likelihood of capturing a micro-objectin the trap 132.

In some embodiments, dielectrophoretic (DEP) forces are applied acrossthe fluidic medium 180 (e.g., in the flow path and/or in thesequestration pens) via one or more electrodes (not shown) tomanipulate, transport, separate and sort micro-objects located therein.For example, in some embodiments, DEP forces are applied to one or moreportions of microfluidic circuit 120 in order to transfer a singlemicro-object from the flow path 106 into a desired microfluidicsequestration pen. In some embodiments, DEP forces are used to prevent amicro-object within a sequestration pen (e.g., sequestration pen 124,126, 128, or 130) from being displaced therefrom. Further, in someembodiments, DEP forces are used to selectively remove a micro-objectfrom a sequestration pen that was previously collected in accordancewith the teachings of the instant invention. In some embodiments, theDEP forces comprise optoelectronic tweezer (OET) forces.

In other embodiments, optoelectrowetting (OEW) forces are applied to oneor more positions in the support structure 104 (and/or the cover 110) ofthe microfluidic device 100 (e.g., positions helping to define the flowpath and/or the sequestration pens) via one or more electrodes (notshown) to manipulate, transport, separate and sort droplets located inthe microfluidic circuit 120. For example, in some embodiments, OEWforces are applied to one or more positions in the support structure 104(and/or the cover 110) in order to transfer a single droplet from theflow path 106 into a desired microfluidic sequestration pen. In someembodiments, OEW forces are used to prevent a droplet within asequestration pen (e.g., sequestration pen 124, 126, 128, or 130) frombeing displaced therefrom. Further, in some embodiments, OEW forces areused to selectively remove a droplet from a sequestration pen that waspreviously collected in accordance with the teachings of the instantinvention.

In some embodiments, DEP and/or OEW forces are combined with otherforces, such as flow and/or gravitational force, so as to manipulate,transport, separate and sort micro-objects and/or droplets within themicrofluidic circuit 120. For example, the enclosure 102 can be tilted(e.g., by tilting device 190) to position the flow path 106 andmicro-objects located therein above the microfluidic sequestration pens,and the force of gravity can transport the micro-objects and/or dropletsinto the pens. In some embodiments, the DEP and/or OEW forces can beapplied prior to the other forces. In other embodiments, the DEP and/orOEW forces can be applied after the other forces. In still otherinstances, the DEP and/or OEW forces can be applied at the same time asthe other forces or in an alternating manner with the other forces.

FIGS. 2A-2F illustrates various embodiments of microfluidic devices thatcan be used in the practice of the present invention. FIG. 2A depicts anembodiment in which the microfluidic device 200 is configured as anoptically-actuated electrokinetic device. A variety ofoptically-actuated electrokinetic devices are known in the art,including devices having an optoelectronic tweezer (OET) configurationand devices having an opto-electrowetting (OEW) configuration. Examplesof suitable OET configurations are illustrated in the following U.S.patent documents, each of which is incorporated herein by reference inits entirety: U.S. Pat. No. RE 44,711 (Wu et al.) (originally issued asU.S. Pat. No. 7,612,355); and U.S. Pat. No. 7,956,339 (Ohta et al.).Examples of OEW configurations are illustrated in U.S. Pat. No.6,958,132 (Chiou et al.) and U.S. Patent Application Publication No.2012/0024708 (Chiou et al.), both of which are incorporated by referenceherein in their entirety. Yet another example of an optically-actuatedelectrokinetic device includes a combined OET/OEW configuration,examples of which are shown in U.S. Patent Publication Nos. 20150306598(Khandros et al.) and 20150306599 (Khandros et al.) and theircorresponding PCT Publications WO2015/164846 and WO2015/164847, all ofwhich are incorporated herein by reference in their entirety.

Motive Microfluidic Device Configurations.

As described above, the control and monitoring equipment of the systemcan comprise a motive module 162 for selecting and moving objects, suchas micro-objects or droplets, in the microfluidic circuit of amicrofluidic device. The microfluidic device can have a variety ofmotive configurations, depending upon the type of object being moved andother considerations. For example, a dielectrophoresis (DEP)configuration can be utilized to select and move micro-objects in themicrofluidic circuit. Thus, the support structure 104 and/or cover 110of the microfluidic device 100 can comprise a DEP configuration forselectively inducing DEP forces on micro-objects in a fluidic medium 180in the microfluidic circuit 120 and thereby select, capture, and/or moveindividual micro-objects or groups of micro-objects. Alternatively, thesupport structure 104 and/or cover 110 of the microfluidic device 100can comprise an electrowetting (EW) configuration for selectivelyinducing EW forces on droplets in a fluidic medium 180 in themicrofluidic circuit 120 and thereby select, capture, and/or moveindividual droplets or groups of droplets.

One example of a microfluidic device 200 comprising a DEP configurationis illustrated in FIGS. 2A and 2B. While for purposes of simplicityFIGS. 2A and 2B show a side cross-sectional view and a topcross-sectional view, respectively, of a portion of an enclosure 102 ofthe microfluidic device 200 having an open region/chamber 202, it shouldbe understood that the region/chamber 202 may be part of a fluidiccircuit element having a more detailed structure, such as a growthchamber, a sequestration pen, a flow region, or a flow channel.Furthermore, the microfluidic device 200 may include other fluidiccircuit elements. For example, the microfluidic device 200 can include aplurality of growth chambers or sequestration pens and/or one or moreflow regions or flow channels, such as those described herein withrespect to microfluidic device 100. A DEP configuration may beincorporated into any such fluidic circuit elements of the microfluidicdevice 200, or select portions thereof. It should be further appreciatedthat any of the above or below described microfluidic device componentsand system components may be incorporated in and/or used in combinationwith the microfluidic device 200. For example, system 150 includingcontrol and monitoring equipment 152, described above, may be used withmicrofluidic device 200, including one or more of the media module 160,motive module 162, imaging module 164, tilting module 166, and othermodules 168.

As seen in FIG. 2A, the microfluidic device 200 includes a supportstructure 104 having a bottom electrode 204 and an electrode activationsubstrate 206 overlying the bottom electrode 204, and a cover 110 havinga top electrode 210, with the top electrode 210 spaced apart from thebottom electrode 204. The top electrode 210 and the electrode activationsubstrate 206 define opposing surfaces of the region/chamber 202. Amedium 180 contained in the region/chamber 202 thus provides a resistiveconnection between the top electrode 210 and the electrode activationsubstrate 206. A power source 212 configured to be connected to thebottom electrode 204 and the top electrode 210 and create a biasingvoltage between the electrodes, as required for the generation of DEPforces in the region/chamber 202, is also shown. The power source 212can be, for example, an alternating current (AC) power source.

In certain embodiments, the microfluidic device 200 illustrated in FIGS.2A and 2B can have an optically-actuated DEP configuration. Accordingly,changing patterns of light 222 from the light source 220, which may becontrolled by the motive module 162, can selectively activate anddeactivate changing patterns of DEP electrodes at regions 214 of theinner surface 208 of the electrode activation substrate 206.(Hereinafter the regions 214 of a microfluidic device having a DEPconfiguration are referred to as “DEP electrode regions.”) Asillustrated in FIG. 2B, a light pattern 222 directed onto the innersurface 208 of the electrode activation substrate 206 can illuminateselect DEP electrode regions 214 a (shown in white) in a pattern, suchas a square. The non-illuminated DEP electrode regions 214(cross-hatched) are hereinafter referred to as “dark” DEP electroderegions 214. The relative electrical impedance through the DEP electrodeactivation substrate 206 (i.e., from the bottom electrode 204 up to theinner surface 208 of the electrode activation substrate 206 whichinterfaces with the medium 180 in the flow region 106) is greater thanthe relative electrical impedance through the medium 180 in theregion/chamber 202 (i.e., from the inner surface 208 of the electrodeactivation substrate 206 to the top electrode 210 of the cover 110) ateach dark DEP electrode region 214. An illuminated DEP electrode region214 a, however, exhibits a reduced relative impedance through theelectrode activation substrate 206 that is less than the relativeimpedance through the medium 180 in the region/chamber 202 at eachilluminated DEP electrode region 214 a.

With the power source 212 activated, the foregoing DEP configurationcreates an electric field gradient in the fluidic medium 180 betweenilluminated DEP electrode regions 214 a and adjacent dark DEP electroderegions 214, which in turn creates local DEP forces that attract orrepel nearby micro-objects (not shown) in the fluidic medium 180. DEPelectrodes that attract or repel micro-objects in the fluidic medium 180can thus be selectively activated and deactivated at many different suchDEP electrode regions 214 at the inner surface 208 of the region/chamber202 by changing light patterns 222 projected from a light source 220into the microfluidic device 200. Whether the DEP forces attract orrepel nearby micro-objects can depend on such parameters as thefrequency of the power source 212 and the dielectric properties of themedium 180 and/or micro-objects (not shown).

The square pattern 224 of illuminated DEP electrode regions 214 aillustrated in FIG. 2B is an example only. Any pattern of the DEPelectrode regions 214 can be illuminated (and thereby activated) by thepattern of light 222 projected into the device 200, and the pattern ofilluminated/activated DEP electrode regions 214 can be repeatedlychanged by changing or moving the light pattern 222.

In some embodiments, the electrode activation substrate 206 can compriseor consist of a photoconductive material. In such embodiments, the innersurface 208 of the electrode activation substrate 206 can befeatureless. For example, the electrode activation substrate 206 cancomprise or consist of a layer of hydrogenated amorphous silicon(a-Si:H). The a-Si:H can comprise, for example, about 8% to 40% hydrogen(calculated as 100*(the number of hydrogen atoms)/(the total number ofhydrogen and silicon atoms)). The layer of a-Si:H can have a thicknessof about 500 nm to about 2.0 In such embodiments, the DEP electroderegions 214 can be created anywhere and in any pattern on the innersurface 208 of the electrode activation substrate 208, in accordancewith the light pattern 222. The number and pattern of the DEP electroderegions 214 thus need not be fixed, but can correspond to the lightpattern 222. Examples of microfluidic devices having a DEP configurationcomprising a photoconductive layer such as discussed above have beendescribed, for example, in U.S. Pat. No. RE 44,711 (Wu et al.)(originally issued as U.S. Pat. No. 7,612,355), the entire contents ofwhich are incorporated herein by reference.

In other embodiments, the electrode activation substrate 206 cancomprise a substrate comprising a plurality of doped layers,electrically insulating layers (or regions), and electrically conductivelayers that form semiconductor integrated circuits, such as is known insemiconductor fields. For example, the electrode activation substrate206 can comprise a plurality of phototransistors, including, forexample, lateral bipolar phototransistors, each phototransistorcorresponding to a DEP electrode region 214. Alternatively, theelectrode activation substrate 206 can comprise electrodes (e.g.,conductive metal electrodes) controlled by phototransistor switches,with each such electrode corresponding to a DEP electrode region 214.The electrode activation substrate 206 can include a pattern of suchphototransistors or phototransistor-controlled electrodes. The pattern,for example, can be an array of substantially square phototransistors orphototransistor-controlled electrodes arranged in rows and columns, suchas shown in FIG. 2B. Alternatively, the pattern can be an array ofsubstantially hexagonal phototransistors or phototransistor-controlledelectrodes that form a hexagonal lattice. Regardless of the pattern,electric circuit elements can form electrical connections between theDEP electrode regions 214 at the inner surface 208 of the electrodeactivation substrate 206 and the bottom electrode 210, and thoseelectrical connections (i.e., phototransistors or electrodes) can beselectively activated and deactivated by the light pattern 222. When notactivated, each electrical connection can have high impedance such thatthe relative impedance through the electrode activation substrate 206(i.e., from the bottom electrode 204 to the inner surface 208 of theelectrode activation substrate 206 which interfaces with the medium 180in the region/chamber 202) is greater than the relative impedancethrough the medium 180 (i.e., from the inner surface 208 of theelectrode activation substrate 206 to the top electrode 210 of the cover110) at the corresponding DEP electrode region 214. When activated bylight in the light pattern 222, however, the relative impedance throughthe electrode activation substrate 206 is less than the relativeimpedance through the medium 180 at each illuminated DEP electroderegion 214, thereby activating the DEP electrode at the correspondingDEP electrode region 214 as discussed above. DEP electrodes that attractor repel micro-objects (not shown) in the medium 180 can thus beselectively activated and deactivated at many different DEP electroderegions 214 at the inner surface 208 of the electrode activationsubstrate 206 in the region/chamber 202 in a manner determined by thelight pattern 222.

Examples of microfluidic devices having electrode activation substratesthat comprise phototransistors have been described, for example, in U.S.Pat. No. 7,956,339 (Ohta et al.) (see, e.g., device 300 illustrated inFIGS. 21 and 22, and descriptions thereof), the entire contents of whichare incorporated herein by reference. Examples of microfluidic deviceshaving electrode activation substrates that comprise electrodescontrolled by phototransistor switches have been described, for example,in U.S. Patent Publication No. 2014/0124370 (Short et al.) (see, e.g.,devices 200, 400, 500, 600, and 900 illustrated throughout the drawings,and descriptions thereof), the entire contents of which are incorporatedherein by reference.

In some embodiments of a DEP configured microfluidic device, the topelectrode 210 is part of a first wall (or cover 110) of the enclosure102, and the electrode activation substrate 206 and bottom electrode 204are part of a second wall (or support structure 104) of the enclosure102. The region/chamber 202 can be between the first wall and the secondwall. In other embodiments, the electrode 210 is part of the second wall(or support structure 104) and one or both of the electrode activationsubstrate 206 and/or the electrode 210 are part of the first wall (orcover 110). Moreover, the light source 220 can alternatively be used toilluminate the enclosure 102 from below.

With the microfluidic device 200 of FIGS. 2A-2B having a DEPconfiguration, the motive module 162 can select a micro-object (notshown) in the medium 180 in the region/chamber 202 by projecting a lightpattern 222 into the device 200 to activate a first set of one or moreDEP electrodes at DEP electrode regions 214 a of the inner surface 208of the electrode activation substrate 206 in a pattern (e.g., squarepattern 224) that surrounds and captures the micro-object. The motivemodule 162 can then move the captured micro-object by moving the lightpattern 222 relative to the device 200 to activate a second set of oneor more DEP electrodes at DEP electrode regions 214. Alternatively, thedevice 200 can be moved relative to the light pattern 222.

In other embodiments, the microfluidic device 200 can have a DEPconfiguration that does not rely upon light activation of DEP electrodesat the inner surface 208 of the electrode activation substrate 206. Forexample, the electrode activation substrate 206 can comprise selectivelyaddressable and energizable electrodes positioned opposite to a surfaceincluding at least one electrode (e.g., cover 110). Switches (e.g.,transistor switches in a semiconductor substrate) may be selectivelyopened and closed to activate or inactivate DEP electrodes at DEPelectrode regions 214, thereby creating a net DEP force on amicro-object (not shown) in region/chamber 202 in the vicinity of theactivated DEP electrodes. Depending on such characteristics as thefrequency of the power source 212 and the dielectric properties of themedium (not shown) and/or micro-objects in the region/chamber 202, theDEP force can attract or repel a nearby micro-object. By selectivelyactivating and deactivating a set of DEP electrodes (e.g., at a set ofDEP electrodes regions 214 that forms a square pattern 224), one or moremicro-objects in region/chamber 202 can be trapped and moved within theregion/chamber 202. The motive module 162 in FIG. 1 can control suchswitches and thus activate and deactivate individual ones of the DEPelectrodes to select, trap, and move particular micro-objects (notshown) around the region/chamber 202. Microfluidic devices having a DEPconfiguration that includes selectively addressable and energizableelectrodes are known in the art and have been described, for example, inU.S. Pat. No. 6,294,063 (Becker et al.) and U.S. Pat. No. 6,942,776(Medoro), the entire contents of which are incorporated herein byreference.

As yet another example, the microfluidic device 200 can have anelectrowetting (EW) configuration, which can be in place of the DEPconfiguration or can be located in a portion of the microfluidic device200 that is separate from the portion which has the DEP configuration.The EW configuration can be an opto-electrowetting configuration or anelectrowetting on dielectric (EWOD) configuration, both of which areknown in the art. In some EW configurations, the support structure 104has an electrode activation substrate 206 sandwiched between adielectric layer (not shown) and the bottom electrode 204. Thedielectric layer can comprise a hydrophobic material and/or can becoated with a hydrophobic material. For microfluidic devices 200 thathave an EW configuration, the inner surface 208 of the support structure104 is the inner surface of the dielectric layer or its hydrophobiccoating.

The dielectric layer (not shown) can comprise one or more oxide layers,and can have a thickness of about 50 nm to about 250 nm (e.g., about 125nm to about 175 nm). In certain embodiments, the dielectric layer maycomprise a layer of oxide, such as a metal oxide (e.g., aluminum oxideor hafnium oxide). In certain embodiments, the dielectric layer cancomprise a dielectric material other than a metal oxide, such as siliconoxide or a nitride. Regardless of the exact composition and thickness,the dielectric layer can have an impedance of about 10 kOhms to about 50kOhms.

In some embodiments, the surface of the dielectric layer that facesinward toward region/chamber 202 is coated with a hydrophobic material.The hydrophobic material can comprise, for example, fluorinated carbonmolecules. Examples of fluorinated carbon molecules includeperfluoro-polymers such as polytetrafluoroethylene (e.g., TEFLON®) orpoly(2,3-difluoromethylenyl-perfluorotetrahydrofuran) (e.g., CYTOP™).Molecules that make up the hydrophobic material can be covalently bondedto the surface of the dielectric layer. For example, molecules of thehydrophobic material can be covalently bound to the surface of thedielectric layer by means of a linker, such as a siloxane group, aphosphonic acid group, or a thiol group. Thus, in some embodiments, thehydrophobic material can comprise alkyl-terminated siloxane,alkyl-termination phosphonic acid, or alkyl-terminated thiol. The alkylgroup can be long-chain hydrocarbons (e.g., having a chain of at least10 carbons, or at least 16, 18, 20, 22, or more carbons). Alternatively,fluorinated (or perfluorinated) carbon chains can be used in place ofthe alkyl groups. Thus, for example, the hydrophobic material cancomprise fluoroalkyl-terminated siloxane, fluoroalkyl-terminatedphosphonic acid, or fluoroalkyl-terminated thiol. In some embodiments,the hydrophobic coating has a thickness of about 10 nm to about 50 nm.In other embodiments, the hydrophobic coating has a thickness of lessthan 10 nm (e.g., less than 5 nm, or about 1.5 to 3.0 nm).

In some embodiments, the cover 110 of a microfluidic device 200 havingan electrowetting configuration is coated with a hydrophobic material(not shown) as well. The hydrophobic material can be the samehydrophobic material used to coat the dielectric layer of the supportstructure 104, and the hydrophobic coating can have a thickness that issubstantially the same as the thickness of the hydrophobic coating onthe dielectric layer of the support structure 104. Moreover, the cover110 can comprise an electrode activation substrate 206 sandwichedbetween a dielectric layer and the top electrode 210, in the manner ofthe support structure 104. The electrode activation substrate 206 andthe dielectric layer of the cover 110 can have the same compositionand/or dimensions as the electrode activation substrate 206 and thedielectric layer of the support structure 104. Thus, the microfluidicdevice 200 can have two eletrowetting surfaces.

In some embodiments, the electrode activation substrate 206 can comprisea photoconductive material, such as described above. Accordingly, incertain embodiments, the electrode activation substrate 206 can compriseor consist of a layer of hydrogenated amorphous silicon (a-Si:H). Thea-Si:H can comprise, for example, about 8% to 40% hydrogen (calculatedas 100*(the number of hydrogen atoms)/(the total number of hydrogen andsilicon atoms)). The layer of a-Si:H can have a thickness of about 500nm to about 2.0 μm. Alternatively, the electrode activation substrate206 can comprise electrodes (e.g., conductive metal electrodes)controlled by phototransistor switches, as described above. Microfluidicdevices having an opto-electrowetting configuration are known in the artand/or can be constructed with electrode activation substrates known inthe art. For example, U.S. Pat. No. 6,958,132 (Chiou et al.), the entirecontents of which are incorporated herein by reference, disclosesopto-electrowetting configurations having a photoconductive materialsuch as a-Si:H, while U.S. Patent Publication No. 2014/0124370 (Short etal.), referenced above, discloses electrode activation substrates havingelectrodes controlled by phototransistor switches.

The microfluidic device 200 thus can have an opto-electrowettingconfiguration, and light patterns 222 can be used to activatephotoconductive EW regions or photoresponsive EW electrodes in theelectrode activation substrate 206. Such activated EW regions or EWelectrodes of the electrode activation substrate 206 can generate anelectrowetting force at the inner surface 208 of the support structure104 (i.e., the inner surface of the overlaying dielectric layer or itshydrophobic coating). By changing the light patterns 222 (or movingmicrofluidic device 200 relative to the light source 220) incident onthe electrode activation substrate 206, droplets (e.g., containing anaqueous medium, solution, or solvent) contacting the inner surface 208of the support structure 104 can be moved through an immiscible fluid(e.g., an oil medium) present in the region/chamber 202.

In other embodiments, microfluidic devices 200 can have an EWODconfiguration, and the electrode activation substrate 206 can compriseselectively addressable and energizable electrodes that do not rely uponlight for activation. The electrode activation substrate 206 thus caninclude a pattern of such electrowetting (EW) electrodes. The pattern,for example, can be an array of substantially square EW electrodesarranged in rows and columns, such as shown in FIG. 2B. Alternatively,the pattern can be an array of substantially hexagonal EW electrodesthat form a hexagonal lattice. Regardless of the pattern, the EWelectrodes can be selectively activated (or deactivated) by electricalswitches (e.g., transistor switches in a semiconductor substrate). Byselectively activating and deactivating EW electrodes in the electrodeactivation substrate 206, droplets (not shown) contacting the innersurface 208 of the overlaying dielectric layer or its hydrophobiccoating can be moved within the region/chamber 202. The motive module162 in FIG. 1 can control such switches and thus activate and deactivateindividual EW electrodes to select and move particular droplets aroundregion/chamber 202. Microfluidic devices having a EWOD configurationwith selectively addressable and energizable electrodes are known in theart and have been described, for example, in U.S. Pat. No. 8,685,344(Sundarsan et al.), the entire contents of which are incorporated hereinby reference.

Regardless of the configuration of the microfluidic device 200, a powersource 212 can be used to provide a potential (e.g., an AC voltagepotential) that powers the electrical circuits of the microfluidicdevice 200. The power source 212 can be the same as, or a component of,the power source 192 referenced in FIG. 1. Power source 212 can beconfigured to provide an AC voltage and/or current to the top electrode210 and the bottom electrode 204. For an AC voltage, the power source212 can provide a frequency range and an average or peak power (e.g.,voltage or current) range sufficient to generate net DEP forces (orelectrowetting forces) strong enough to trap and move individualmicro-objects (not shown) in the region/chamber 202, as discussed above,and/or to change the wetting properties of the inner surface 208 of thesupport structure 104 (i.e., the dielectric layer and/or the hydrophobiccoating on the dielectric layer) in the region/chamber 202, as alsodiscussed above. Such frequency ranges and average or peak power rangesare known in the art. See, e.g., U.S. Pat. No. 6,958,132 (Chiou et al.),U.S. Pat. No. RE44,711 (Wu et al.) (originally issued as U.S. Pat. No.7,612,355), and US Patent Application Publication Nos. US2014/0124370(Short et al.), US2015/0306598 (Khandros et al.), and US2015/0306599(Khandros et al.).

Sequestration Pens.

Non-limiting examples of generic sequestration pens 244, 246, and 248are shown within the microfluidic device 240 depicted in FIGS. 2C and2D. Each sequestration pen 244, 246, and 248 can comprise an isolationstructure 250 defining an isolation region 258 and a connection region254 fluidically connecting the isolation region 258 to a channel 122.The connection region 254 can comprise a proximal opening 252 to thechannel 122 and a distal opening 256 to the isolation region 258. Theconnection region 254 can be configured so that the maximum penetrationdepth of a flow of a fluidic medium (not shown) flowing from the channel122 into the sequestration pen 244, 246, 248 does not extend into theisolation region 258. Thus, due to the connection region 254, amicro-object (not shown) or other material (not shown) disposed in anisolation region 258 of a sequestration pen 244, 246, 248 can thus beisolated from, and not substantially affected by, a flow of medium 180in the channel 122.

The channel 122 can thus be an example of a swept region, and theisolation regions 258 of the sequestration pens 244, 246, 248 can beexamples of unswept regions. As noted, the channel 122 and sequestrationpens 244, 246, 248 can be configured to contain one or more fluidicmedia 180. In the example shown in FIGS. 2C-2D, the ports 242 areconnected to the channel 122 and allow a fluidic medium 180 to beintroduced into or removed from the microfluidic device 240. Once themicrofluidic device 240 contains the fluidic medium 180, the flow 260 offluidic medium 180 in the channel 122 can be selectively generated andstopped. For example, as shown, the ports 242 can be disposed atdifferent locations (e.g., opposite ends) of the channel 122, and a flow260 of medium can be created from one port 242 functioning as an inletto another port 242 functioning as an outlet.

FIG. 2E illustrates a detailed view of an example of a sequestration pen244 according to the present invention. Examples of micro-objects 270are also shown.

As is known, a flow 260 of fluidic medium 180 in a microfluidic channel122 past a proximal opening 252 of sequestration pen 244 can cause asecondary flow 262 of the medium 180 into and/or out of thesequestration pen 244. To isolate micro-objects 270 in the isolationregion 258 of a sequestration pen 244 from the secondary flow 262, thelength L_(con) of the connection region 254 of the sequestration pen 244(i.e., from the proximal opening 252 to the distal opening 256) shouldbe greater than the penetration depth D_(p) of the secondary flow 262into the connection region 254. The penetration depth D_(p) of thesecondary flow 262 depends upon the velocity of the fluidic medium 180flowing in the channel 122 and various parameters relating to theconfiguration of the channel 122 and the proximal opening 252 of theconnection region 254 to the channel 122. For a given microfluidicdevice, the configurations of the channel 122 and the opening 252 willbe fixed, whereas the rate of flow 260 of fluidic medium 180 in thechannel 122 will be variable. Accordingly, for each sequestration pen244, a maximal velocity V_(max) for the flow 260 of fluidic medium 180in channel 122 can be identified that ensures that the penetration depthD_(p) of the secondary flow 262 does not exceed the length L_(con) ofthe connection region 254. As long as the rate of the flow 260 offluidic medium 180 in the channel 122 does not exceed the maximumvelocity V_(max), the resulting secondary flow 262 can be limited to thechannel 122 and the connection region 254 and kept out of the isolationregion 258. The flow 260 of medium 180 in the channel 122 will thus notdraw micro-objects 270 out of the isolation region 258. Rather,micro-objects 270 located in the isolation region 258 will stay in theisolation region 258 regardless of the flow 260 of fluidic medium 180 inthe channel 122.

Moreover, as long as the rate of flow 260 of medium 180 in the channel122 does not exceed V_(max), the flow 260 of fluidic medium 180 in thechannel 122 will not move miscellaneous particles (e.g., microparticlesand/or nanoparticles) from the channel 122 into the isolation region 258of a sequestration pen 244. Having the length L_(con) of the connectionregion 254 be greater than the maximum penetration depth D_(p) of thesecondary flow 262 can thus prevent contamination of one sequestrationpen 244 with miscellaneous particles from the channel 122 or anothersequestration pen (e.g., sequestration pens 246, 248 in FIG. 2D).

Because the channel 122 and the connection regions 254 of thesequestration pens 244, 246, 248 can be affected by the flow 260 ofmedium 180 in the channel 122, the channel 122 and connection regions254 can be deemed swept (or flow) regions of the microfluidic device240. The isolation regions 258 of the sequestration pens 244, 246, 248,on the other hand, can be deemed unswept (or non-flow) regions. Forexample, components (not shown) in a first fluidic medium 180 in thechannel 122 can mix with a second fluidic medium 280 in the isolationregion 258 substantially only by diffusion of components of the firstmedium 180 from the channel 122 through the connection region 254 andinto the second fluidic medium 280 in the isolation region 258.Similarly, components (not shown) of the second medium 280 in theisolation region 258 can mix with the first medium 180 in the channel122 substantially only by diffusion of components of the second medium280 from the isolation region 258 through the connection region 254 andinto the first medium 180 in the channel 122. The first medium 180 canbe the same medium or a different medium than the second medium 280.Moreover, the first medium 180 and the second medium 280 can start outbeing the same, then become different (e.g., through conditioning of thesecond medium 280 by one or more cells in the isolation region 258, orby changing the medium 180 flowing through the channel 122).

The maximum penetration depth D_(p) of the secondary flow 262 caused bythe flow 260 of fluidic medium 180 in the channel 122 can depend on anumber of parameters, as mentioned above. Examples of such parametersinclude: the shape of the channel 122 (e.g., the channel can directmedium into the connection region 254, divert medium away from theconnection region 254, or direct medium in a direction substantiallyperpendicular to the proximal opening 252 of the connection region 254to the channel 122); a width W_(ch) (or cross-sectional area) of thechannel 122 at the proximal opening 252; and a width W_(con) (orcross-sectional area) of the connection region 254 at the proximalopening 252; the velocity V of the flow 260 of fluidic medium 180 in thechannel 122; the viscosity of the first medium 180 and/or the secondmedium 280, or the like.

In some embodiments, the dimensions of the channel 122 and sequestrationpens 244, 246, 248 can be oriented as follows with respect to the vectorof the flow 260 of fluidic medium 180 in the channel 122: the channelwidth W_(ch) (or cross-sectional area of the channel 122) can besubstantially perpendicular to the flow 260 of medium 180; the widthW_(con) (or cross-sectional area) of the connection region 254 atopening 252 can be substantially parallel to the flow 260 of medium 180in the channel 122; and/or the length L_(con) of the connection regioncan be substantially perpendicular to the flow 260 of medium 180 in thechannel 122. The foregoing are examples only, and the relative positionof the channel 122 and sequestration pens 244, 246, 248 can be in otherorientations with respect to each other.

As illustrated in FIG. 2E, the width W_(con) of the connection region254 can be uniform from the proximal opening 252 to the distal opening256. The width W_(con) of the connection region 254 at the distalopening 256 can thus be in any of the ranges identified herein for thewidth W_(con) of the connection region 254 at the proximal opening 252.Alternatively, the width W_(con) of the connection region 254 at thedistal opening 256 can be larger than the width W_(con) of theconnection region 254 at the proximal opening 252.

As illustrated in FIG. 2E, the width of the isolation region 258 at thedistal opening 256 can be substantially the same as the width W_(con) ofthe connection region 254 at the proximal opening 252. The width of theisolation region 258 at the distal opening 256 can thus be in any of theranges identified herein for the width W_(con) of the connection region254 at the proximal opening 252. Alternatively, the width of theisolation region 258 at the distal opening 256 can be larger or smallerthan the width W_(con) of the connection region 254 at the proximalopening 252. Moreover, the distal opening 256 may be smaller than theproximal opening 252 and the width W_(con) of the connection region 254may be narrowed between the proximal opening 252 and distal opening 256.For example, the connection region 254 may be narrowed between theproximal opening and the distal opening, using a variety of differentgeometries (e.g. chamfering the connection region, beveling theconnection region). Further, any part or subpart of the connectionregion 254 may be narrowed (e.g. a portion of the connection regionadjacent to the proximal opening 252).

In various embodiments of sequestration pens (e.g. 124, 126, 128, 130,244, 246 or 248), the isolation region (e.g. 258) is configured tocontain a plurality of micro-objects. In other embodiments, theisolation region can be configured to contain only one, two, three,four, five, or a similar relatively small number of micro-objects.Accordingly, the volume of an isolation region can be, for example, atleast 3×10³, 6×10³, 9×10³, 1×10⁴, 2×10⁴, 4×10⁴, 8×10⁴, 1×10⁵, 2×10⁵,4×10⁵, 8×10⁵, 1×10⁶, 2×10⁶, 4×10⁶, 6×10⁶ cubic microns, or more.

In various embodiments of sequestration pens, the width W_(ch) of thechannel 122 at a proximal opening (e.g. 252) can be within any of thefollowing ranges: 50-1000 microns, 50-500 microns, 50-400 microns,50-300 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100microns, 70-500 microns, 70-400 microns, 70-300 microns, 70-250 microns,70-200 microns, 70-150 microns, 90-400 microns, 90-300 microns, 90-250microns, 90-200 microns, 90-150 microns, 100-300 microns, 100-250microns, 100-200 microns, 100-150 microns, and 100-120 microns. Theforegoing are examples only, and the width W_(ch) of the channel 122 canbe in other ranges (e.g., a range defined by any of the endpoints listedabove). Moreover, the W_(ch) of the channel 122 can be selected to be inany of these ranges in regions of the channel other than at a proximalopening of a sequestration pen.

In some embodiments, a sequestration pen has a cross-sectional height ofabout 30 to about 200 microns, or about 50 to about 150 microns. In someembodiments, the sequestration pen has a cross-sectional area of about100,000 to about 2,500,000 square microns, or about 200,000 to about2,000,000 square microns. In some embodiments, a connection region has across-sectional height that matches the cross-sectional height of thecorresponding sequestration pen. In some embodiments, the connectionregion has a cross-sectional width of about 50 to about 500 microns, orabout 100 to about 300 microns.

In various embodiments of sequestration pens the height H_(ch) of thechannel 122 at a proximal opening 252 can be within any of the followingranges: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns,20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns,40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50microns. The foregoing are examples only, and the height H_(ch) of thechannel 122 can be in other ranges (e.g., a range defined by any of theendpoints listed above). The height H_(ch) of the channel 122 can beselected to be in any of these ranges in regions of the channel otherthan at a proximal opening of a sequestration pen.

In various embodiments of sequestration pens a cross-sectional area ofthe channel 122 at a proximal opening 252 can be within any of thefollowing ranges: 500-50,000 square microns, 500-40,000 square microns,500-30,000 square microns, 500-25,000 square microns, 500-20,000 squaremicrons, 500-15,000 square microns, 500-10,000 square microns, 500-7,500square microns, 500-5,000 square microns, 1,000-25,000 square microns,1,000-20,000 square microns, 1,000-15,000 square microns, 1,000-10,000square microns, 1,000-7,500 square microns, 1,000-5,000 square microns,2,000-20,000 square microns, 2,000-15,000 square microns, 2,000-10,000square microns, 2,000-7,500 square microns, 2,000-6,000 square microns,3,000-20,000 square microns, 3,000-15,000 square microns, 3,000-10,000square microns, 3,000-7,500 square microns, or 3,000 to 6,000 squaremicrons. The foregoing are examples only, and the cross-sectional areaof the channel 122 at a proximal opening 252 can be in other ranges(e.g., a range defined by any of the endpoints listed above).

In various embodiments of sequestration pens, the length L_(con) of theconnection region 254 can be in any of the following ranges: 1-200microns, 5-150 microns, 10-100 microns, 15-80 microns, 20-60 microns,20-500 microns, 40-400 microns, 60-300 microns, 80-200 microns, and100-150 microns. The foregoing are examples only, and length L_(con) ofa connection region 254 can be in a different ranges than the foregoingexamples (e.g., a range defined by any of the endpoints listed above).

In various embodiments of sequestration pens the width W_(con) of aconnection region 254 at a proximal opening 252 can be in any of thefollowing ranges: 20-500 microns, 20-400 microns, 20-300 microns, 20-200microns, 20-150 microns, 20-100 microns, 20-80 microns, 20-60 microns,30-400 microns, 30-300 microns, 30-200 microns, 30-150 microns, 30-100microns, 30-80 microns, 30-60 microns, 40-300 microns, 40-200 microns,40-150 microns, 40-100 microns, 40-80 microns, 40-60 microns, 50-250microns, 50-200 microns, 50-150 microns, 50-100 microns, 50-80 microns,60-200 microns, 60-150 microns, 60-100 microns, 60-80 microns, 70-150microns, 70-100 microns, and 80-100 microns. The foregoing are examplesonly, and the width W_(con) of a connection region 254 at a proximalopening 252 can be different than the foregoing examples (e.g., a rangedefined by any of the endpoints listed above).

In various embodiments of sequestration pens the width W_(con) of aconnection region 254 at a proximal opening 252 can be in any of thefollowing ranges: 2-35 microns, 2-25 microns, 2-20 microns, 2-15microns, 2-10 microns, 2-7 microns, 2-5 microns, 2-3 microns, 3-25microns, 3-20 microns, 3-15 microns, 3-10 microns, 3-7 microns, 3-5microns, 3-4 microns, 4-20 microns, 4-15 microns, 4-10 microns, 4-7microns, 4-5 microns, 5-15 microns, 5-10 microns, 5-7 microns, 6-15microns, 6-10 microns, 6-7 microns, 7-15 microns, 7-10 microns, 8-15microns, and 8-10 microns. The foregoing are examples only, and thewidth W_(con) of a connection region 254 at a proximal opening 252 canbe different than the foregoing examples (e.g., a range defined by anyof the endpoints listed above).

In various embodiments of sequestration pens, a ratio of the lengthL_(con) of a connection region 254 to a width W_(con) of the connectionregion 254 at the proximal opening 252 can be greater than or equal toany of the following ratios: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or more. The foregoing are examplesonly, and the ratio of the length L_(con) of a connection region 254 toa width W_(con) of the connection region 254 at the proximal opening 252can be different than the foregoing examples.

In various embodiments of microfluidic devices 100, 200, 240, 290,V_(max) can be set around 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,1.1, 1.2, 1.3, 1.4, or 1.5 μL/sec.

In various embodiments of microfluidic devices having sequestrationpens, the volume of an isolation region 258 of a sequestration pen canbe, for example, at least 3×10³, 6×10³, 9×10³, 1×10⁴, 2×10⁴, 4×10⁴,8×10⁴, 1×10⁵, 2×10⁵, 4×10⁵, 8×10⁵, 1×10⁶, 2×10⁶, 4×10⁶, 6×10⁶ cubicmicrons, or more. In various embodiments of microfluidic devices havingsequestration pens, the volume of a sequestration pen may be about5×10³, 7×10³, 1×10⁴, 3×10⁴, 5×10⁴, 8×10⁴, 1×10⁵, 2×10⁵, 4×10⁵, 6×10⁵,8×10⁵, 1×10⁶, 2×10⁶, 4×10⁶, 8×10⁶, 1×10⁷, 3×10⁷, 5×10⁷, or about 8×10⁷cubic microns, or more. In some embodiments, the microfluidic device hassequestration pens wherein no more than 1×10² biological cells may bemaintained, and the volume of a sequestration pen may be no more than2×10⁶ cubic microns. In some embodiments, the microfluidic device hassequestration pens wherein no more than 1×10² biological cells may bemaintained, and a sequestration pen may be no more than 4×10⁵ cubicmicrons. In yet other embodiments, the microfluidic device hassequestration pens wherein no more than 50 biological cells may bemaintained, a sequestration pen may be no more than 4×10⁵ cubic microns.

In various embodiment, the microfluidic device has sequestration pensconfigured as in any of the embodiments discussed herein where themicrofluidic device has about 100 to about 500 sequestration pens; about200 to about 1000 sequestration pens, about 500 to about 1500sequestration pens, about 1000 to about 2000 sequestration pens, orabout 1000 to about 3500 sequestration pens.

In some other embodiments, the microfluidic device has sequestrationpens configured as in any of the embodiments discussed herein where themicrofluidic device has about 1500 to about 3000 sequestration pens,about 2000 to about 3500 sequestration pens, about 2500 to about 4000sequestration pens, about 3000 to about 4500 sequestration pens, about3500 to about 5000 sequestration pens, about 4000 to about 5500sequestration pens, about 4500 to about 6000 sequestration pens, about5000 to about 6500 sequestration pens, about 5500 to about 7000sequestration pens, about 6000 to about 7500 sequestration pens, about6500 to about 8000 sequestration pens, about 7000 to about 8500sequestration pens, about 7500 to about 9000 sequestration pens, about8000 to about 9500 sequestration pens, about 8500 to about 10,000sequestration pens, about 9000 to about 10,500 sequestration pens, about9500 to about 11,000 sequestration pens, about 10,000 to about 11,500sequestration pens, about 10,500 to about 12,000 sequestration pens,about 11,000 to about 12,500 sequestration pens, about 11,500 to about13,000 sequestration pens, about 12,000 to about 13,500 sequestrationpens, about 12,500 to about 14,000 sequestration pens, about 13,000 toabout 14,500 sequestration pens, about 13,500 to about 15,000sequestration pens, about 14,000 to about 15,500 sequestration pens,about 14,500 to about 16,000 sequestration pens, about 15,000 to about16,500 sequestration pens, about 15,500 to about 17,000 sequestrationpens, about 16,000 to about 17,500 sequestration pens, about 16,500 toabout 18,000 sequestration pens, about 17,000 to about 18,500sequestration pens, about 17,500 to about 19,000 sequestration pens,about 18,000 to about 19,500 sequestration pens, about 18,500 to about20,000 sequestration pens, about 19,000 to about 20,500 sequestrationpens, about 19,500 to about 21,000 sequestration pens, or about 20,000to about 21,500 sequestration pens.

FIG. 2F illustrates a microfluidic device 290 according to oneembodiment. The microfluidic device 290 is illustrated in FIG. 2F is astylized diagram of a microfluidic device 100. In practice themicrofluidic device 290 and its constituent circuit elements (e.g.channels 122 and sequestration pens 128) would have the dimensionsdiscussed herein. The microfluidic circuit 120 illustrated in FIG. 2Fhas two ports 107, four distinct channels 122 and four distinct flowpaths 106. The microfluidic device 290 further comprises a plurality ofsequestration pens opening off of each channel 122. In the microfluidicdevice illustrated in FIG. 2F, the sequestration pens have a geometrysimilar to the pens illustrated in FIG. 2E and thus, have bothconnection regions and isolation regions. Accordingly, the microfluidiccircuit 120 includes both swept regions (e.g. channels 122 and portionsof the connection regions 254 within the maximum penetration depth D_(p)of the secondary flow 262) and non-swept regions (e.g. isolation regions258 and portions of the connection regions 254 not within the maximumpenetration depth D_(p) of the secondary flow 262).

FIGS. 3A through 3D shows various embodiments of system 150 which can beused to operate and observe microfluidic devices (e.g. 100, 200, 240,290) according to the present invention. As illustrated in FIG. 3A, thesystem 150 can include a structure (“nest”) 300 configured to hold amicrofluidic device 100 (not shown), or any other microfluidic devicedescribed herein. The nest 300 can include a socket 302 capable ofinterfacing with the microfluidic device 360 (e.g., anoptically-actuated electrokinetic device 100) and providing electricalconnections from power source 192 to microfluidic device 360. The nest300 can further include an integrated electrical signal generationsubsystem 304. The electrical signal generation subsystem 304 can beconfigured to supply a biasing voltage to socket 302 such that thebiasing voltage is applied across a pair of electrodes in themicrofluidic device 360 when it is being held by socket 302. Thus, theelectrical signal generation subsystem 304 can be part of power source192. The ability to apply a biasing voltage to microfluidic device 360does not mean that a biasing voltage will be applied at all times whenthe microfluidic device 360 is held by the socket 302. Rather, in mostcases, the biasing voltage will be applied intermittently, e.g., only asneeded to facilitate the generation of electrokinetic forces, such asdielectrophoresis or electro-wetting, in the microfluidic device 360.

As illustrated in FIG. 3A, the nest 300 can include a printed circuitboard assembly (PCBA) 320. The electrical signal generation subsystem304 can be mounted on and electrically integrated into the PCBA 320. Theexemplary support includes socket 302 mounted on PCBA 320, as well.

Typically, the electrical signal generation subsystem 304 will include awaveform generator (not shown). The electrical signal generationsubsystem 304 can further include an oscilloscope (not shown) and/or awaveform amplification circuit (not shown) configured to amplify awaveform received from the waveform generator. The oscilloscope, ifpresent, can be configured to measure the waveform supplied to themicrofluidic device 360 held by the socket 302. In certain embodiments,the oscilloscope measures the waveform at a location proximal to themicrofluidic device 360 (and distal to the waveform generator), thusensuring greater accuracy in measuring the waveform actually applied tothe device. Data obtained from the oscilloscope measurement can be, forexample, provided as feedback to the waveform generator, and thewaveform generator can be configured to adjust its output based on suchfeedback. An example of a suitable combined waveform generator andoscilloscope is the Red Pitaya™.

In certain embodiments, the nest 300 further comprises a controller 308,such as a microprocessor used to sense and/or control the electricalsignal generation subsystem 304. Examples of suitable microprocessorsinclude the Arduino™ microprocessors, such as the Arduino Nano™. Thecontroller 308 may be used to perform functions and analysis or maycommunicate with an external master controller 154 (shown in FIG. 1) toperform functions and analysis. In the embodiment illustrated in FIG. 3Athe controller 308 communicates with a master controller 154 through aninterface 310 (e.g., a plug or connector).

In some embodiments, the nest 300 can comprise an electrical signalgeneration subsystem 304 comprising a Red Pitaya™ waveformgenerator/oscilloscope unit (“Red Pitaya™ unit”) and a waveformamplification circuit that amplifies the waveform generated by the RedPitaya™ unit and passes the amplified voltage to the microfluidic device100. In some embodiments, the Red Pitaya™ unit is configured to measurethe amplified voltage at the microfluidic device 360 and then adjust itsown output voltage as needed such that the measured voltage at themicrofluidic device 360 is the desired value. In some embodiments, thewaveform amplification circuit can have a +6.5V to −6.5V power supplygenerated by a pair of DC-DC converters mounted on the PCBA 320,resulting in a signal of up to 13 Vpp at the microfluidic device 360.

As illustrated in FIG. 3A, the nest 300 can further include a thermalcontrol subsystem 306. The thermal control subsystem 306 can beconfigured to regulate the temperature of microfluidic device 360 heldby the support structure 300. For example, the thermal control subsystem306 can include a Peltier thermoelectric device (not shown) and acooling unit (not shown). The Peltier thermoelectric device can have afirst surface configured to interface with at least one surface of themicrofluidic device 360. The cooling unit can be, for example, a coolingblock (not shown), such as a liquid-cooled aluminum block. A secondsurface of the Peltier thermoelectric device (e.g., a surface oppositethe first surface) can be configured to interface with a surface of sucha cooling block. The cooling block can be connected to a fluidic path330 configured to circulate cooled fluid through the cooling block. Inthe embodiment illustrated in FIG. 3A, the support structure 300comprises an inlet 332 and an outlet 334 to receive cooled fluid from anexternal reservoir (not shown), introduce the cooled fluid into thefluidic path 330 and through the cooling block, and then return thecooled fluid to the external reservoir. In some embodiments, the Peltierthermoelectric device, the cooling unit, and/or the fluidic path 330 canbe mounted on a casing 340 of the support structure 300. In someembodiments, the thermal control subsystem 306 is configured to regulatethe temperature of the Peltier thermoelectric device so as to achieve atarget temperature for the microfluidic device 360. Temperatureregulation of the Peltier thermoelectric device can be achieved, forexample, by a thermoelectric power supply, such as a Pololu™thermoelectric power supply (Pololu Robotics and Electronics Corp.). Thethermal control subsystem 306 can include a feedback circuit, such as atemperature value provided by an analog circuit. Alternatively, thefeedback circuit can be provided by a digital circuit.

In some embodiments, the nest 300 can include a thermal controlsubsystem 306 with a feedback circuit that is an analog voltage dividercircuit (shown in FIG. 3B) which includes a resistor (e.g., withresistance 1 kOhm+/−0.1%, temperature coefficient +/−0.02 ppm/C0) and aNTC thermistor (e.g., with nominal resistance 1 kOhm+/−0.01%). In someinstances, the thermal control subsystem 306 measures the voltage fromthe feedback circuit and then uses the calculated temperature value asinput to an on-board PID control loop algorithm. Output from the PIDcontrol loop algorithm can drive, for example, both a directional and apulse-width-modulated signal pin on a Pololu™ motor drive (not shown) toactuate the thermoelectric power supply, thereby controlling the Peltierthermoelectric device.

The nest 300 can include a serial port 350 which allows themicroprocessor of the controller 308 to communicate with an externalmaster controller 154 via the interface 310. In addition, themicroprocessor of the controller 308 can communicate (e.g., via a Plinktool (not shown)) with the electrical signal generation subsystem 304and thermal control subsystem 306. Thus, via the combination of thecontroller 308, the interface 310, and the serial port 350, theelectrical signal generation subsystem 308 and the thermal controlsubsystem 306 can communicate with the external master controller 154.In this manner, the master controller 154 can, among other things,assist the electrical signal generation subsystem 308 by performingscaling calculations for output voltage adjustments. A Graphical UserInterface (GUI), one example of which is shown in FIG. 3C, provided viaa display device 170 coupled to the external master controller 154, canbe configured to plot temperature and waveform data obtained from thethermal control subsystem 306 and the electrical signal generationsubsystem 308, respectively. Alternatively, or in addition, the GUI canallow for updates to the controller 308, the thermal control subsystem306, and the electrical signal generation subsystem 304.

As discussed above, system 150 can include an imaging device 194. Insome embodiments, the imaging device 194 comprises a light modulatingsubsystem 404. The light modulating subsystem 404 can include a digitalmirror device (DMD) or a microshutter array system (MSA), either ofwhich can be configured to receive light from a light source 402 andtransmits a subset of the received light into an optical train ofmicroscope 400. Alternatively, the light modulating subsystem 404 caninclude a device that produces its own light (and thus dispenses withthe need for a light source 402), such as an organic light emittingdiode display (OLED), a liquid crystal on silicon (LCOS) device, aferroelectric liquid crystal on silicon device (FLCOS), or atransmissive liquid crystal display (LCD). The light modulatingsubsystem 404 can be, for example, a projector. Thus, the lightmodulating subsystem 404 can be capable of emitting both structured andunstructured light. One example of a suitable light modulating subsystem404 is the Mosaic™ system from Andor Technologies™. In certainembodiments, imaging module 164 and/or motive module 162 of system 150can control the light modulating subsystem 404.

In certain embodiments, the imaging device 194 further comprises amicroscope 400. In such embodiments, the nest 300 and light modulatingsubsystem 404 can be individually configured to be mounted on themicroscope 400. The microscope 400 can be, for example, a standardresearch-grade light microscope or fluorescence microscope. Thus, thenest 300 can be configured to be mounted on the stage 410 of themicroscope 400 and/or the light modulating subsystem 404 can beconfigured to mount on a port of microscope 400. In other embodiments,the nest 300 and the light modulating subsystem 404 described herein canbe integral components of microscope 400.

In certain embodiments, the microscope 400 can further include one ormore detectors 422. In some embodiments, the detector 422 is controlledby the imaging module 164. The detector 422 can include an eye piece, acharge-coupled device (CCD), a camera (e.g., a digital camera), or anycombination thereof. If at least two detectors 422 are present, onedetector can be, for example, a fast-frame-rate camera while the otherdetector can be a high sensitivity camera. Furthermore, the microscope400 can include an optical train configured to receive reflected and/oremitted light from the microfluidic device 360 and focus at least aportion of the reflected and/or emitted light on the one or moredetectors 422. The optical train of the microscope can also includedifferent tube lenses (not shown) for the different detectors, such thatthe final magnification on each detector can be different.

In certain embodiments, imaging device 194 is configured to use at leasttwo light sources. For example, a first light source 402 can be used toproduce structured light (e.g., via the light modulating subsystem 404)and a second light source 432 can be used to provide unstructured light.The first light source 402 can produce structured light foroptically-actuated electrokinesis and/or fluorescent excitation, and thesecond light source 432 can be used to provide bright fieldillumination. In these embodiments, the motive module 162 can be used tocontrol the first light source 404 and the imaging module 164 can beused to control the second light source 432. The optical train of themicroscope 400 can be configured to (1) receive structured light fromthe light modulating subsystem 404 and focus the structured light on atleast a first region in a microfluidic device, such as anoptically-actuated electrokinetic device, when the device is being heldby the support structure 200, and (2) receive reflected and/or emittedlight from the microfluidic device and focus at least a portion of suchreflected and/or emitted light onto detector 422. The optical train canbe further configured to receive unstructured light from a second lightsource and focus the unstructured light on at least a second region ofthe microfluidic device, when the device is held by the supportstructure 300. In certain embodiments, the first and second regions ofthe microfluidic device can be overlapping regions. For example, thefirst region can be a subset of the second region.

In FIG. 3D, the first light source 402 is shown supplying light to alight modulating subsystem 404, which provides structured light to theoptical train of the microscope 400. The second light source 432 isshown providing unstructured light to the optical train via a beamsplitter 436. Structured light from the light modulating subsystem 404and unstructured light from the second light source 432 travel from thebeam splitter 436 through the optical train together to reach a secondbeam splitter (or dichroic filter 406, depending on the light providedby the light modulating subsystem 404) where the light gets reflecteddown through the objective 408 to the sample plane 412. Reflected and/oremitted light from the sample plane 412 then travels back up through theobjective 408, through the beam splitter and/or dichroic filter 406, andto another dichroic filter 424. Only a fraction of the light reachingdichroic filter 424 passes through and reaches the detector 422.

In some embodiments, the second light source 432 emits blue light. Withan appropriate dichroic filter 424, blue light reflected from the sampleplane 412 is able to pass through dichroic filter 424 and reach thedetector 422. In contrast, structured light coming from the lightmodulating subsystem 404 gets reflected from the sample plane 412, butdoes not pass through the dichroic filter 424. In this example, thedichroic filter 424 is filtering out visible light having a wavelengthlonger than 495 nm. Such filtering out of the light from the lightmodulating subsystem 404 would only be complete (as shown) if the lightemitted from the light modulating subsystem did not include anywavelengths shorter than 495 nm. In practice, if the light coming fromthe light modulating subsystem 404 includes wavelengths shorter than 495nm (e.g., blue wavelengths), then some of the light from the lightmodulating subsystem would pass through filter 424 to reach the detector422. In such an embodiment, the filter 424 acts to change the balancebetween the amount of light that reaches the detector 422 from the firstlight source 402 and the second light source 432. This can be beneficialif the first light source 402 is significantly stronger than the secondlight source 432. In other embodiments, the second light source 432 canemit red light, and the dichroic filter 424 can filter out visible lightother than red light (e.g., visible light having a wavelength shorterthan 650 nm).

FIG. 3E illustrates communications between the motive module 164 and thelight modulating subsystem 404 to project patterns of light on amicrofluidic device according to a specific embodiment of the invention.As discussed above with respect to FIG. 3D, the light modulatingsubsystem 404 may comprise an electrically-addressed spatial lightmodulator and/or an optically-addressed spatial light modulator.Electrically-addressed spatial light modulators comprise an array ofindividually-addressable spatial light modulators (i.e. spatial lightmodulating elements) that are controlled by electrodes. In FIG. 3E, thelight modulating subsystem 404 is a Digital Mirror Device (DMD) 460comprising an array of individually-addressable micro-mirrors 464 thatare controlled by an electrodes. However, in other embodiments, thelight modulating subsystem 404 can be a Liquid Crystal on Silicon (LCoS)device comprising an array of individually-addressable electrodes thatcorrespond to pixels in a liquid crystal display.

In the embodiment illustrated in FIG. 3E, the light modulating subsystem404 uses a separate light source 440 to receive and modulate light.However, in other embodiments, the light modulating subsystem 404comprises its own light source.

As illustrated in FIG. 3E, the motive module 162 transmits information450 specifying a specific pattern of light (“pattern information”) tothe light modulating subsystem 404. In some embodiments, the patterninformation 450 can comprise a bitmap (or similar pixel-based datastructure), vector data, or any combination thereof. For purposes ofillustration, the pattern information 450 in FIG. 3E is illustrated as abitmap comprising an array of pixels 454 and including a square pattern452 of pixels. Depending on the embodiment, the pattern information 450can be binary (i.e. specify whether or not to project a pattern oflight) or contain values indicating an intensity of light to project. Ininstances where the spatial light modulators are micro-mirrors 464, themicro-mirrors 464 may create different intensities of light by rapidlyswitching the mirrors between an “on” and “off” state (i.e. “dithering”the micro-mirrors).

The light modulating subsystem 404 receives the pattern information 450from the motive module 162 and uses the pattern information 450 todirect the projection of a pattern of light 468 onto DEP electroderegions 474 on the microfluidic device 470. In the embodimentillustrated in FIG. 3E, a DMD 460 rotates a plurality 462 ofindividually-addressable micro-mirrors 464 corresponding to the squarepattern information 450 into an “on state.” The square pattern ofindividual-addressable micro-mirrors 462 modulates the light from thelight source 440 to project a pattern of light 468 onto the microfluidicdevice 470 that illuminates a square pattern of DEP electrode regions472 in the array of DEP electrode regions 474 in the microfluidic device470.

In some embodiments, there is a one-to-one correspondence between thearray of individually-addressable spatial light modulating elements 464that project light onto the microfluidic device 470 and the array of DEPelectrode regions 474 in the microfluidic device 470. In this way, eachindividually-addressable spatial light modulating element 464 canproject light to generate light-actuated DEP force at a correspondingDEP electrode region 474. In these embodiments, the motive module 162can send pattern information 450 to the light modulating subsystem 404that specifies the DEP electrode regions 474 to project light onto. Forexample, instead of sending bitmap and or vector data to the lightmodulating subsystem 404, the motive module 162 can communicate directlywith the individually-addressable spatial light modulators to controlwhich of the DEP electrode regions 474 are illuminated on themicrofluidic device 470. Once illuminated the DEP electrode regions 474may exert OET or OEW force on surrounding micro-objects.

As discussed above, in some embodiments, the spatial light modulatingelements 464 can receive pattern information 450 specifying an intensityof light to project. In a specific embodiment, the pattern information450 may specify a gradation of light to project over adjacent DEPelectrode regions 474 in the microfluidic device. In some embodiments,the pattern information 450 may specify a gradation of light thatdecreases in intensity over adjacent DEP electrode regions 474. Forexample, the pattern information 450 may specify that about 100% of themaximum light intensity is to be projected at a first DEP electroderegion 474, that 70% of the maximum light intensity is to be projectedat a second DEP electrode region 474 adjacent to the first DEP electroderegion 474, and that 10% of the maximum light intensity is to beprojected at a third DEP electrode region 474 adjacent to the second DEPelectrode region 474. Various combinations of light intensities may beused to project a gradation over various numbers of DEP electroderegions 474 (e.g. any decreasing combination of about 100%, about 90%,about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about20%, and about 10%, and any values therebetween, of the maximum lightintensity over any number of DEP electrode regions 474 and). Similarly,the pattern information 450 may specify a gradation of light thatincreases in intensity over any number of DEP electrode regions 474 or agradation of light that both increases and decreases in intensity overany number of DEP electrode regions 474.

In one aspect, the invention provides methods for the automateddetection of a micro-object of interest disposed within a microfluidicdevice. The micro-object of interest can be a cell, such as a mammaliancell (e.g., a blood cell, a hybridoma, a cancer cell, a transformedcell, or the like). Alternatively, the micro-object of interest can be abead, such as might be used in an assay (e.g., a microbead, a magneticbead, or the like).

More specifically, the invention provides methods of automaticallydetecting a micro-object of interest that has similar morphology tofeatures of the microfluidic device. In some instances, detection ofmicro-objects disposed within a microfluidic device can be complicatedby other features of the microfluidic device that have similarmorphology to the micro-object of interest. For example, in instanceswhere cells have a diameter of 10 microns, it may be difficult todistinguish the cells from a phototransistor array that is a 10 micronby 10 micro grid. In addition, micro-objects such as cells can berelatively translucent compared to various features of the microfluidicdevice. Accordingly, it is necessary to identify and remove unwantedfeatures of the microfluidic device (e.g. photo transistor arrays, wallsor circuit elements of the microfluidic device) prior to identifyingmicro-objects of interest.

The invention provides methods of generating differential and filteredimages that substantially remove features of the microfluidic device(also referred to herein as “microfluidic device features”), butmaintain the micro-objects of interest. In some embodiments, adifferential image is used to remove features of the microfluidicdevice. In these embodiments, a first and second image of themicrofluidic device are taken and used to create a differential image.In certain embodiments, the first and second images are digital images.For example, the first and second images can be captured using a digitalimaging device, such as a digital camera or a CCD device. Alternatively,the first and second images can be obtained in a non-digitized formatand then converted into digital images. After being captured (anddigitized, if necessary), the first and second images can be stored in adigital memory device.

In certain instances, actions may be taken to induce movement of thefluid in the microfluidic device after creating the first image andprior to creating the second image. In these instances, inducingmovement of the fluid present in the region can involve inducing asmall, controlled flow of fluid into or out of the microfluidic device.For example, a pump can be used to introduce a discrete volume of fluid(e.g., 30 pL, 60 pL, 90 pL, or the like) into the flow path of themicrofluidic device, thereby causing all of the fluid in the flow path(and any micro-objects contained within the fluid) to shift a smalldistance (e.g., 2, 5, 10, 15, 20, 30 microns, etc.) in the direction ofthe fluid flow. Alternatively, a pump can be used to suck a discretevolume of fluid (e.g., 30 pL, 60 pL, 90 pL, etc.) from the flow path ofthe microfluidic device, thereby causing all of the fluid in the flowpath (and any micro-objects contained within the fluid) to shift a smalldistance (e.g., 2, 5, 10, 15, 20, 30 microns, etc.) in the direction ofthe fluid flow. In another alternative, the valves that connect themicrofluidic device to a source of fluid can be opened. This typicallyresults in a slight movement of fluid within the microfluidic device,causing fluid in the flow path (and any micro-objects contained withinthe fluid) to shift a small distance. Without intending to be bound bytheory, it is believed that this slight movement is caused by a changein surface tension.

In other instances, the position of the microfluidic device is shiftedrelative to the imaging device 194 after creating the first image andprior to creating the second image. In certain embodiments, shifting theposition of the microfluidic device can involve moving a stage that isholding the microfluidic device. The stage can be part of the imagingdevice 194, such as a conventional microscope (e.g., a light microscopeor fluorescence microscope) or a system suitable for operatingelectrokinetic microfluidic devices. In certain embodiments, themicrofluidic device can be shifted by at least 1, 2, 3 microns, or more.Typically, the shift will be in a direction perpendicular to the opticalaxis of the imaging device 194. For example, if the optical axiscorresponds to the z-axis, the shift of the microfluidic device can bein the x,y plane. However, in some embodiments, the shift can include(or even be limited to) movement along the z-axis. In certainembodiments, a piezoelectric device is used to shift the microfluidicdevice or a stage that is holding the microfluidic device.

After the first image and the second image have been created, variousmethods can be used to further analyze and process the first and secondimages. For example, the light intensity value L_(i) of each pixel P_(i)(i=1 to n) in the image can be evaluated and/or recorded, where n is thenumber of pixels in the image. The light intensity value L_(i) can bethe actual observed light intensity value L_(i,obs) for pixel P_(i).Alternatively, the light intensity values can be smoothed. For example,L_(i) can be an average of the L_(i,obs) for pixel P_(i) and some or allof the pixels that contact (i.e., immediately surround) pixel P_(i). Inanother alternative, the light intensity value L_(i) can be the actualobserved light intensity value L_(i,obs) for pixel P_(i) minus abackground light intensity value L_(bkgd). In certain embodiments, thelight intensity value L_(i) of each pixel P_(i) is represented using 0-8bits, 0-10 bits, 0-12 bits, or 0-14 bits. Using a larger number of bitsto represent the light intensity values L_(i) provides for superioranalysis of weak signals.

In some embodiments, the differential image is generated by subtractingthe first image from the second image. In other embodiments, thedifferential image is generated by subtracting the second image from thefirst image. In some embodiments, a negative light intensity value L_(i)may be assigned to some or all of the pixels P_(i) from one of theimages and a positive light intensity value L_(i) may be assigned tosome or all of the pixels P_(i) from the other image. In theseembodiments, the light intensity values L_(i) for the pixels P_(i) inthe two images may be added to subtract one image from the other image.By subtracting the same pixels present in the first image and the secondimage, static features of the microfluidic device, as opposed to mobilemicro-objects of interest, can be removed prior to analyzing the imageto identify the micro-objects of interest. For example, thephototransistor array and/or microfluidic circuit elements (e.g. walls)can be removed by creating a differential image that subtracts outpixels that represent such features.

Prior to subtracting one image from the other, the first and secondimages can be aligned computationally and regions of the first andsecond images that can't be aligned can optionally be discard. Suchalignment can involve the use of one or more identifiable referencepoints, such as circuit elements within the microfluidic device (e.g. achannel or a sequestration pen within the microfluidic device). Thedifferential image can be generated computationally and then stored in adigital memory device.

After a differential image has been created, various methods can be usedto further analyze and process the differential image prior toidentifying micro-objects of interest. In certain embodiments, analyzingthe differential image further comprises identifying each pixel P_(i)that has a positive light intensity value L_(i) as being apositive-value pixel and identifying each pixel P_(i) that has anegative light intensity value L_(i) as a negative-value pixel. In otherembodiments, analyzing the differential image further comprises:comparing the light intensity value L_(i) of each pixel P_(i) to apredetermined threshold light intensity value L^(o); and identifyingeach pixel P_(i) having an L_(i) greater than L^(o) as a positive-valuepixel and each pixel P_(i) having an L_(i) less than −1*L^(o) as anegative-value pixel. In certain embodiments, L^(o) can be based on theaverage light intensity value L_(avg) of the set of light intensityvalues L_(i) obtained from the set of pixels P_(i) (i=1 to n). For thecalculation of L_(avg), any pixel P_(i) having a light intensity valueL_(i) that is negative can be multiplied by the factor −1 prior to thecalculation. Thus, for example, L^(o) can be equal to L_(avg). Incertain related embodiments, L^(o) can be based on the average lightintensity value L_(avg) and the standard deviation σ of the set of lightintensity values L_(i) obtained from the set of pixels P_(i) (i=1 to n).For example, L^(o) can equal L_(avg) plus some multiple of σ (e.g.,L^(o) can equal L_(avg)+1.6σ, L_(avg)+2.0σ, L_(avg)+3.0σ, etc.). Incertain embodiments, an optimum value for L^(o) is determinedempirically.

In alternate embodiments, computational transforms may be used toidentify and remove microfluidic device features from one or more imagesof the microfluidic device. For example, a Fourier transform may be usedto identify features of the microfluidic device that are periodic, suchas a phototransistor array, periodic circuit elements (e.g. walls) andfilter out the periodic features. In a specific embodiment, a DiscreteFourier Transform (“DFT”) is applied to an image of the microfluidicdevice and pixels corresponding to the frequency domain of the DFT arefiltered out of the image. After the pixels corresponding to thefrequency domain are filtered out, an inverse DFT is applied to producea filtered image. In embodiments where a filtered image is created, someor all pixels P_(i) in the image have positive light intensity valuesL_(i) and can be processed and/or analyzed as discussed above with therespect to differential images.

In certain embodiments, a single pixel can corresponds to an area in themicrofluidic device that is substantially similar to the cross-sectionalarea of a micro-object of interest. Each pixel can, for example,correspond to an area in the microfluidic device of substantially 5microns² (or 4 microns², 3 microns², 2 microns², 1 microns², etc.) andthe cross-sectional area of a micro-object can be substantially 5microns² (or 4 microns², 3 microns², 2 microns², 1 microns², etc.). Insuch embodiments, a single positive-value (or negative value) pixel canrepresent the location of a micro-object. Thus, in embodiments that usea differential image to identify micro-objects of interest, pixelsidentified as positive-value pixels can represent the current locationof a micro-object (i.e., the position after movement of the fluid), andpixels identified as negative-value pixels can represent the formerlocation of a micro-object (i.e., the position before movement of thefluid). However, if the differential image is generated by subtractingthe second image from the first image, then pixels identified aspositive-value pixels can represent the former location of amicro-object and pixels identified as negative-value pixels canrepresent the current location of the micro-object.

In other embodiments, a single pixel can correspond to an area in themicrofluidic device that is substantially smaller than thecross-sectional area of a micro-object of interest. For example, themicro-object may have a cross-sectional area of about 80 microns²,whereas a pixel may correspond to an area of about 2 microns². In suchembodiments, one or more clusters of pixels will be required to coverthe cross-sectional area of the micro-object (e.g., in the foregoingexample, it would take substantially 40 pixels to cover thecross-section area of the micro-object, or 24 pixels to cover thecross-sectional area of the circumference of the micro-object).Accordingly, in certain embodiments, analyzing the differential and/orfiltered image further comprises determining whether a set ofpositive-value (or negative-value) pixels form one or more clusters ofpixels (i.e. pixel clusters) aggregated proximal to each other andwhether the area represented by the set of pixel cluster(s) issufficiently large in comparison to the micro-object being detected. Forexample, a set of pixel clusters that correspond to an area that is atleast 50% of (or 60%, 70%, 80%, 90%, or substantially similar to) thecross-sectional area of a micro-object of interest can be identified asrepresenting the location of the micro-object. Alternatively, a set ofpixel clusters that correspond to an area that is at least 70% of (or80%, 90%, or substantially similar to) the cross-sectional circumferenceof a micro-object of interest can be identified as representing thelocation of the micro-object. In instances, where the images includemultiple micro-objects, multiple sets of pixel clusters can beidentified and analyzed to determine whether each set of pixel clusterscorresponds to a micro-object of interest.

The analysis of a set of pixel clusters can further comprise a number ofother features aside from the area and circumference of the pixelclusters. The set of pixel clusters may be analyzed according to globalmorphology (i.e. the size and shape of the set of one or more pixelclusters), local morphology (i.e. the size and shape of the individualpixel clusters), positive and negative light intensity values L_(i), andother features based on a combination of these elements (e.g. lightintensity as a function of size). Various methods may be used to analyzethe set of pixel clusters including traditional machine learningtechniques where the above-discussed features are computed for a set ofimages of micro-objects and used to train a classifier to identifymicro-objects of interest in new images based on the same features.

In certain embodiments, the differential image can be analyzed for pairsof positive-value and negative-value pixels (or sets of pixel clusters),and the identification of a micro-object can be limited to instances inwhich the differential image contains corresponding positive-value and anegative-value pixel (or sets of pixel clusters). In certain relatedembodiments, the identification of a micro-object can be limited toinstances in which the differential image contains a pair ofpositive-value and negative-value pixels (or sets of pixel clusters) andthe difference in the relative positions of the positive-value andnegative-value pixels (or sets of pixel clusters) is consistent with themagnitude of movement of fluid induced in the region.

In addition to information specifying whether a micro-object is present,micro-object identification can provide various additional information.As discussed above, the differential and/or filtered image may beanalyzed with respect to the size and shape of the potentialmicro-object. In doing so, various information regarding themicro-object may be produced including the radius of the micro-object,the perimeter of the micro-object and a centroid associated with themicro-object.

Once the micro-objects have been identified, various operations can beperformed on the micro-objects. In some embodiments, the cellidentification will be used to count micro-objects in the microfluidiccircuit. In some embodiments, the identified micro-objects will beassociated with various circuit elements of the microfluidic circuit(e.g. channel, a sequestration pen, a trap, or any combination thereof)and/or spatial locations on the microfluidic circuit. In theseembodiments, the density of micro-objects in a specific area of themicrofluidic circuit (e.g. a channel, a sequestration pen, a trap, orany combination thereof) may be calculated.

Micro-object identification may also be used in conjunction withmanipulating or repositioning the micro-objects using force, such as OETor DEP force. In some embodiments, micro-objects that are identified ina specific circuit element (e.g. channel or sequestration pen) orlocation of the microfluidic circuit may be moved to (i.e. repositionedin) another type of circuit element or location of the microfluidiccircuit. For example, micro-objects may be identified in a channel inthe microfluidic circuit and repositioned in sequestration pens in themicrofluidic circuit (referred to herein as “penning” a micro-object).Conversely, micro-objects identified in sequestration pens in themicrofluidic circuit may be moved to in channels in the microfluidiccircuit. Alternately, one or more micro-objects may identified in onesequestration pen and repositioned in an empty sequestration pen(referred to herein as “re-penning” a micro-object). According to theembodiment, the micro-objects may be moved using various mechanisms,including OET and DEP force. Similarly, micro-objects may berepositioned sequentially (i.e. one micro-object at a time), inparallel, or any combination thereof (e.g. sequentially repositioninggroups of multiple cells in parallel).

In instances where micro-objects are repositioned from the channel toindividual sequestration pens (or re-penning from an individualsequestration pen to another sequestration pen), different algorithmsmay be used to assign micro-objects to empty sequestration pens. In someembodiments, an algorithm will be used to assign micro-objects to emptysequestration pens such that distance between the micro-objects and thepens (i.e. the trajectory or path that the micro-objects have to travelduring repositioning) is minimized. In these embodiments, the use offorce (e.g. OET or DEP force) to move the micro-objects is alsominimized because the micro-objects are only required to travel aminimum distance to be repositioned in an empty sequestration pen.

In these embodiments, a local micro-object density in a channel (i.e.number of micro-objects within a specific spatial area of the channel)may be used to determine a suitable algorithm to assign specificmicro-objects in the channel to empty sequestration pens. Localmicro-object density may be computed in a number of ways. In someembodiments, local micro-object density may be computed based on a fixedsize area (e.g. 200 microns², or an area of the channel 100 microns longand extending the width of the channel) or using approaches that usevarious sizes of areas. In other embodiments, local micro-object densitymay calculated based on clusters of identified micro-objects or thedistance between identified micro-objects. Local micro-object densityalso may be computed by subdividing the channel into a grid or using a“sliding window” approach to compute density for overlapping areas ofthe channel.

If the local micro-object density is above a threshold value T1_(density), then micro-objects may be assigned to the nearest emptysequestration pens such that the distance between the micro-objects andsequestration pens is minimized. If the local micro-object density isbelow a specific threshold value T1 _(density), then the emptysequestration pens may be assigned to the micro-objects that are closestto the empty sequestration pens, such that the distance between themicro-objects and the sequestration pens is minimized. In someinstances, local T1 _(density), may be computed based on the number ofempty pens as well as the density of micro-objects within the channel ina predefined neighborhood area.

Different methods of computing the distance between a micro-object andan empty sequestration pen (i.e. the trajectory the micro-object or pathneeds to be moved during penning) may be used to assign specificmicro-objects to empty sequestration pens. In some embodiments, thedistance between the micro-object and a potential sequestration pen maybe computed based only on the optimal trajectory using OET and/or DEPforce. In some instances, the optimal trajectory using OET or DEP forceinvolves a combination of orthogonal motion paths (e.g. combination ofdistinct movement only along a y-axis and an x-axis) to move themicro-objects. In other instances, the distance may be based on theshortest possible path between the micro-object and the sequestrationpen, without constraint (i.e. the micro-objects may travel along anypath to reach the sequestration pens). In most embodiments, themicro-objects will be re-positioned (i.e. “penned” or “re-penned”) usingthe same trajectory as determined by the algorithm used to calculate thedistance (trajectory).

Similarly, in instances where a large number of micro-objects areassigned to sequestration pens (or vice versa), different algorithms maybe used to compute the optimal assignment of micro-objects to pens (orvice versa). These algorithms can use different computational methods ofdetermining a micro-object-to-sequestration pen assignment thatminimizes the overall distance (i.e. length of the trajectory) that themicro-objects need to be moved in order to reposition the micro-objectsinto sequestration pens. For example, the algorithms may use the sum ofthe lengths of all the trajectories as a heuristic to minimize thedistance that the micro-objects need to travel. In some embodiments,constraints such as a maximum distance that a micro-object can be movedduring repositioning may be introduced into the computation of theoptimal assignment. Various combinatorial algorithms may be used tocompute the optimal assignment between micro-objects and sequestrationpens. Suitable algorithms include: greedy algorithms, nonlinearoptimization, heuristic-based algorithms and constrained search. Othersimilar algorithms are known in the art.

Once the optimal assignment and trajectory has been computed for themicro-objects, a force, such as OET and/or DEP, may be used to move themicro-objects to their assigned pens. The micro-objects may berepositioned using patterns of light, such as a “light cage”, thatsurround the micro-objects and subject the micro-objects to OET and/orDEP force or by using bars or similar structures to apply OET and/or DEPforce to the micro-objects. Typically, a light cage will be a structurethat substantially encloses the micro-object (e.g. a square, a circle ora polygon). However, in some instances, a light cage may contain a breakor an opening such that the micro-object is not fully enclosed.

As discussed above, in most embodiments, the micro-objects will be movedaccording to the distance (trajectory) used to compute the optimalassignment of micro-objects to pens. According to the embodiment,micro-objects may be moved sequentially or in parallel any combinationthereof (e.g. sequentially moving groups of cells in parallel). Inembodiments where the micro-objects are moved in parallel, the algorithmused to compute the optimal assignment or trajectory may compare thetrajectories and ensure that the micro-objects do not collide when theyare moved in parallel by modifying the trajectory and assignments of themicro-objects to pens. In a specific embodiment, the algorithm may“swap” micro-object assignments to pens when a potential collision isidentified. In this embodiment, when the optimal trajectory for a firstmicro-objects intersects with the optimal trajectory for a secondmicro-objects, the optimal trajectory for the first micro-object isassigned to the second micro-object and the optimal trajectory for thesecond micro-object is assigned to the first micro-object. In anotherspecific embodiment, the algorithm delays the repositioning of the firstmicro-object until such a time that the first and second micro-objectscan move along their respective trajectories without colliding.

In some instances, the micro-object density may be so high that themicro-objects need to be separated from one another prior to assigningthe micro-objects to sequestration pens and repositioning (i.e.“penning” or “re-penning”) the micro-objects. For example, themicro-object density may be so high that the micro-objects cannot bepenned using OET and/or DEP force because the light cage used toreposition objects using OET and/or DEP force cannot be used on a singlemicro-object without interfering with other micro-objects. Thisinterference is of particular concern in instances where it is importantto minimize the amount of OET and/or DEP force applied to themicro-object. For examples, instances where the micro-objects could beharmed by OET and/or DEP force or by-products of OET force (e.g.electrolysis associated with OET and/or DEP force). In these instances,information produced during micro-object identification (e.g. theradius, the centroid, the perimeter and the location of a micro-object)may be used to move the micro-objects such the micro-objects may bepenned or re-penned without interfering with other cells (hereinreferred to as “separating” the micro-objects).

In order to identify instances where the micro-objects need to beseparated prior to penning, a local micro-object density may be computedbased on a defined spatial region and compared to a second thresholdvalue T2 _(density). Alternately, the distance between the micro-objectsmay be computed (e.g. the distance between centroids of micro-objects,the distance between the perimeters of the micro-objects) and used todetermine whether the micro-objects need to be separated. However, ascan appreciated, in some instances, the distance between micro-objectsmay be too small to identify the micro-objects as separate micro-objectsand micro-objects. In these instances, the micro-objects may bere-identified after repositioning (i.e. “penning”) the micro-objects toensure that each sequestration pen contains a single micro-object.

In some embodiments, a modified light box is used to separate themicro-objects prior to, or during, penning (or re-penning). In theseembodiments, a division algorithm is used to compute a set of verticesthat partition each identified micro-object in the spatial region of themicrofluidic device (e.g. the portion of the channel or thesequestration pen) from the other micro-objects in the same spatialregion. However, as can be appreciated by those skilled in the art, theset of vertices may be drawn such that only a subset of themicro-objects in the spatial region of the microfluidic device areseparated from the other micro-objects. For example, the set of verticesmay only separate the subset of micro-objects in the spatial region thatneed to be repositioned due to their close proximity to othermicro-objects.

In a specific embodiment, a Delaunay triangulation is computed using thecentroids of each micro-object. The Delaunay triangulation produces aset of triangles that connect the centroids of the micro-objects. AVoronoi diagram is then computed based on the circumcircles of thetriangles computed using the Delaunay Triangulation. The Voronoi diagramis a set of vertices that divide the spatial area into a sub-areas suchthat the distance between the set of vertices and the centroid of themicro-object is maximized. Other methods of computing a set of verticesthat partition each cell from the other cells in the spatial region areknown in the art.

Once the set of vertices has been computed, the set of vertices can beused in combination with OET and/or DEP forces to move themicro-objects. In one embodiment, one or more “modified light cages” aregenerated using the intersection of set of the vertices and the lightcage shape typically used to move a micro-object (e.g. a square orcircle). Because the intersection of the vertices and the light cagesdefines an area where the light cages do not intersect or overlap, thelight cage can be re-drawn as a modified light cage surrounding theintersection (or a subset of the intersection) such that the modifiedlight cage does not interfere with other micro-objects. The modifiedlight cages can then be used to separate micro-objects by repositioningthe micro-object by moving the micro-objects away from each other. Insome instances, modified light cage may be re-drawn as the micro-objectsare repositioned such that the original light cages are drawn when themicro-objects are in the final position.

Modified light cages may be used to reposition the micro-objects in avariety of embodiments. Depending on the embodiment, the modified lightcages for two proximate micro-objects are used to reposition themicro-objects prior to, or after, computing and selecting the trajectoryand assignment to a sequestration pen for each micro-object. In someembodiments, modified light cages are used to reposition micro-objectsiteratively or sequentially. In addition, modified light cages may beused to pen micro-objects in their assigned sequestration pens. In someembodiments, micro-objects that are closest to the perimeter of thespatial area or closest together in space may be re-positioned or pennedprior to repositioning or penning other micro-objects.

In moving the micro-objects, the speed at which OET and/or DEP is usedto move the cells may be gradually accelerated in order to “ramp up”motion of the micro-objects and ensure that the micro-objects are notlost from their light cages. For example, in a specific embodiment, theinitial velocity of the micro-objects may be gradually accelerated froma low initial velocity to a higher travelling velocity. This gradualacceleration may be applied both in instances where the micro-objectsare automatically repositioned (e.g. penning, re-penning and export) andin instances where the micro-objects are manually repositioned (e.g.manually selecting and moving a cell). Similarly, the high travellingvelocity may be “ramped down” to a final velocity of zero when themicro-objects reach the end of their trajectory and are at their finalposition.

FIGS. 4A, 4B, and 4C illustrate micro-object identification and penningaccording to one embodiment of the invention. In FIG. 4A, biologicalcells within the channel of a microfluidic circuit are shown immediatelyfollowing the identification of the cells and the assignment of cells topens. The black boxes surrounding the cells illustrate the output of thecell identification algorithm—that is, an identification of cellsindicated by a box around the cell. The white boxes surrounding theblack boxes are the light cages of OET force used to reposition thecells. Lastly, the black lines that connect the boxes surrounding thecells to the sequestration pens illustrate the optimal trajectorycomputed in assigning the cells to sequestration pens. FIG. 4B shows thesame cells at a later time point in which the light cages have beenmoved along their selected trajectories. FIG. 4C shows the same cells ata third time point where the light cages have been almost fully movedalong their selected trajectories to position the cells in thesequestration pens.

FIGS. 5A-5F illustrate micro-object separation according to a specificembodiment of the present invention. FIG. 5A illustrates the Delauneytriangulation of a set of micro-objects within a specified spatialregion and the corresponding Voronoi diagram. FIG. 5B illustrates thecorresponding Voronoi diagram without the Delauney triangulation. FIG.5C illustrates light cages typically used to move micro-objects overlaidupon the Voronoi diagram. FIG. 5D illustrates modified light cagesgenerated by computing the intersection between the typical light cagesof FIG. 5C and the Voronoi diagram. FIG. 5E illustrates the separationof the micro-objects that are in close proximity with each other usingthe modified light cages. FIG. 5F illustrates the separatedmicro-objects.

The methods of the invention are useful for the automated detection ofmicro-objects in all types of microfluidic devices. In certainembodiments, the microfluidic device can include a flow region (or flowchannel) and one or more chambers (or sequestration pens).Alternatively, or in addition, the microfluidic device can be anelectrokinetic device, such as an optically actuated electrokineticdevice, or can include a region configured for electrokinesis.Electrokinetic devices, particularly electrokinetic devices having anarray of transistors (e.g., phototransistors), can provide aparticularly complicated background if the transistors in the array havean area that is similar to the cross-sectional area of a micro-objectthat is being detected. The methods described herein can be particularlyeffective at detecting micro-objects disposed in such a device.

In certain embodiments, the invention further provides machine readablestorage devices for storing non-transitory machine readable instructionsfor carrying out any of the methods described herein. The machinereadable instructions can control the imaging device used to obtain theimages and/or a processor (e.g., in a computational device) that alignsthe images, generates differential images, and/or analyzes thedifferential images.

Although specific embodiments and applications of the invention havebeen described in this specification, these embodiments and applicationsare exemplary only, and many variations are possible.

What is claimed:
 1. A method of re-positioning micro-objects in amicrofluidic device, the method comprising: identifying a set ofmicro-objects disposed within a specified spatial region of themicrofluidic device; calculating a set of vertices that divide thespecified spatial region into sub-regions, each of which contains one ormore micro-object(s) of the set of micro-objects; generating a modifiedfirst light cage for at least one micro-object of the set ofmicro-objects based on the calculated set of vertices; and moving themodified first light cage relative to the specified spatial region ofthe microfluidic device to re-position the at least one micro-object. 2.The method of claim 1, wherein calculating said set of vertices thatdivide the specified spatial region into sub-regions comprisescalculating a set of vertices that maximize the distance between asubset of the calculated set of vertices that are adjacent to eachmicro-object of the set of micro-objects and the micro-object.
 3. Themethod of claim 1, wherein calculating said set of vertices comprisescalculating a set of vertices that divide the specified spatial regioninto sub-regions, wherein at least a subset of the sub-regions containsa single micro-object of the set of micro-objects.
 4. The method ofclaim 3, wherein calculating the set of vertices comprises: calculatinga Delaunay triangulation of the set of micro-objects; generating aVoronoi diagram based on the Delaunay triangulation of the set ofmicro-objects; and identifying the set of vertices based on the Voronoidiagram.
 5. The method of claim 1, wherein generating the modified firstlight cage comprises: computing, for a first micro-object of the set ofmicro-objects, a first light cage; computing an intersection between thefirst light cage and the set of vertices; and generating the modifiedfirst light cage based on the intersection between the first light cageand the set of vertices.
 6. The method of claim 5, further comprising:computing, for a second micro-object of the set of micro-objects, asecond light cage; computing the intersection between the second lightcage and the set of vertices; and generating a modified second lightcage based on the intersection between the second light cage and the setof vertices, wherein the modified second light cage does not intersectwith the modified first light cage.
 7. The method of claim 6, furthercomprising moving both the modified first light cage and the modifiedsecond light cage relative to the specified spatial region of themicrofluidic device to physically separate the first micro-object andthe second micro-object.
 8. The method of claim 7, wherein the firstmicro-object and the second micro-object are initially located inadjacent sub-regions of the specified spatial region.
 9. The method ofclaim 1, wherein the at least one micro-object is re-positioned from afirst location in the microfluidic device to a second location.
 10. Themethod of claim 9, wherein the first location is within a microfluidicchannel of the microfluidic device and the second location is within asequestration pen of the microfluidic device.
 11. The method of claim10, wherein the sequestration pen comprises an isolation region and aconnection region which connects the isolation region to themicrofluidic channel.
 12. The method of claim 11, wherein the modifiedfirst light cage moves the at least one micro-object from themicrofluidic channel, through the connection region, and into in theisolation region of the sequestration pen.
 13. The method of claim 9,wherein the first location is within a sequestration pen of themicrofluidic device and the second location is within a microfluidicchannel of the microfluidic device.
 14. The method of claim 9, whereinthe first location is within a first sequestration pen of themicrofluidic device and the second location is within a secondsequestration pen of the microfluidic device.
 15. The method of claim 1,wherein re-positioning the at least one micro-object of the set ofmicro-objects comprises accelerating each of the at least onemicro-objects from an initial velocity to a traveling velocity over afirst time period.
 16. The method of claim 15, wherein re-positioningthe at least one micro-object of the set of micro-objects comprisesdecelerating each of the at least one micro-objects from the travelingvelocity to a final velocity over a second time period.
 17. The methodof claim 1, wherein identifying the set of micro-objects comprises usingmachine learning techniques.
 18. The method of claim 1, wherein the atleast one micro-object is a cell.
 19. The method of claim 18, whereinthe cell is a mammalian cell.
 20. The method of claim 1, wherein themicrofluidic device comprises a dielectrophoresis (DEP) configuration.21. The method of claim 20, wherein the modified first light cageactivates DEP electrodes in the microfluidic device, thereby generatingDEP forces that move the at least one micro-object.
 22. A method ofre-positioning a micro-object in a microfluidic device, the methodcomprising: identifying a set of micro-objects disposed within aspecified spatial region of the microfluidic device; generating amodified first light cage to encompass at least one micro-object of theset of micro-objects; and moving the modified light cage to re-positionthe at least one micro-object and separate it the at least onemicro-object from the set of micro-objects.
 23. The method of claim 22,wherein the at least one micro-object is re-positioned from a firstlocation in the microfluidic device to a second location.
 24. The methodof claim 23, wherein the first location is within a microfluidic channelof the microfluidic device and the second location is within asequestration pen of the microfluidic device.
 25. The method of claim24, wherein the sequestration pen comprises an isolation region and aconnection region which connects the isolation region to themicrofluidic channel.
 26. The method of claim 25, wherein the modifiedfirst light cage moves the at least one micro-object from themicrofluidic channel, through the connection region, and into in theisolation region of the sequestration pen.
 27. The method of claim 23,wherein the first location is within a sequestration pen of themicrofluidic device and the second location is within a microfluidicchannel of the microfluidic device.
 28. The method of claim 23, whereinthe first location is within a first sequestration pen of themicrofluidic device and the second location is within a secondsequestration pen of the microfluidic device.
 29. The method of claim22, wherein re-positioning the at least one micro-object of the set ofmicro-objects comprises accelerating each of the at least onemicro-objects from an initial velocity to a traveling velocity over afirst time period.
 30. The method of claim 29, wherein re-positioningthe at least one micro-object of the set of micro-objects comprisesdecelerating each of the at least one micro-objects from the travelingvelocity to a final velocity over a second time period.
 31. The methodof claim 22, wherein identifying the set of micro-objects comprisesusing machine learning techniques.
 32. The method of claim 22, whereinthe at least one micro-object is a cell.
 33. The method of claim 32,wherein the cell is a mammalian cell.
 34. The method of claim 22,wherein the microfluidic device comprises a dielectrophoresis (DEP)configuration.
 35. The method of claim 34, wherein the modified firstlight cage activates DEP electrodes in the microfluidic device, therebygenerating DEP forces that move the at least one micro-object.